Tricyclic lactams for use in HSPC-sparing treatments for RB-positive abnormal cellular proliferation

ABSTRACT

This invention is in the area of tricyclic lactam compounds for and methods of treating selected Rb-positive cancers and other Rb-positive abnormal cellular proliferative disorders while minimizing the deleterious effects on healthy cells, for example healthy Hematopoietic Stem Cells and Progenitor Cells (HSPCs), associated with current treatment modalities. In one aspect, treatment of select Rb-positive cancers is disclosed using specific compounds disclosed herein. In certain embodiments, the compounds described herein act as selective cyclin-dependent kinase 4/6 (CDK 4/6) inhibitors when administered to subjects.

RELATED APPLICATIONS

This application claims the benefit of provisional U.S. Application No. 61/980,883, filed Apr. 17, 2014, provisional U.S. Application No. 61/980,895, filed Apr. 17, 2014, provisional U.S. Application No. 61/980,918, filed Apr. 17, 2014, and provisional U.S. Application No. 61/980,939, filed Apr. 17, 2014, which are hereby incorporated by reference for all purposes.

GOVERNMENT INTEREST

The U.S. Government has rights in this invention by virtue of support under Grant No. 5R44AI084284 awarded by the National Institute of Allergy and Infectious Diseases.

FIELD

This invention is in the area of tricyclic lactam compounds for and methods of treating selected Rb-positive cancers and other Rb-positive abnormal cellular proliferative disorders while minimizing the deleterious effects on healthy cells, for example healthy Hematopoietic Stem Cells and Progenitor Cells (HSPCs), associated with current treatment modalities.

BACKGROUND

The regulation of the cell cycle is governed and controlled by specific proteins, which are activated and deactivated mainly through phosphorylation/dephosphorylation processes in a precisely timed manner. The key proteins that coordinate the initiation, progression, and completion of cell-cycle program are cyclin dependent kinases (CDKs). Cyclin-dependent kinases belong to the serine-threonine protein kinase family. They are heterodimeric complexes composed of a catalytic kinase subunit and a regulatory cyclin subunit. CDK activity is controlled by association with their corresponding regulatory subunits (cyclins) and CDK inhibitor proteins (Cip & Kip proteins, INK4s), by their phosphorylation state, and by ubiquitin-mediated proteolytic degradation (see D. G. Johnson, C. L. Walker, Annu Rev. Pharmacol. Toxicol 39 (1999) 295-312; D. O. Morgan, Annu Rev. Cell Dev. Biol. 13 (1997) 261-291; C. J. Sherr, Science 274 (1996) 1672-1677; T. Shimamura et al., Bioorg. Med. Chem. Lett. 16 (2006) 3751-3754).

There are four CDKs that are significantly involved in cellular proliferation: CDK1, which predominantly regulates the transition from G2 to M phase, and CDK2, CDK4, and CDK6, which regulate the transition from G1 to S phase (Malumbres M, Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm. Nat. Rev. Cancer 2009; 9(3):153-166). In early to mid G1 phase, when the cell is responsive to mitogenic stimuli, activation of CDK4-cyclin D and CDK6-cyclin D induces phosphorylation of the retinoblastoma protein (pRb). Phosphorylation of pRb releases the transcription factor E2F, which enters the nucleus to activate transcription of other cyclins which promote further progression of the cell cycle (see J. A. Diehl, Cancer Biol. Ther. 1 (2002) 226-231; C. J. Sherr, Cell 73 (1993) 1059-1065). CDK4 and CDK6 are closely related proteins with basically indistinguishable biochemical properties (see M. Malumbres, M. Barbacid, Trends Biochem. Sci. 30 (2005) 630-641).

A number of CDK 4/6 inhibitors have been identified, including specific pyrido[2,3-d]pyrimidines, 2-anilinopyrimidines, diaryl ureas, benzoyl-2,4-diaminothiazoles, indolo[6,7-a]pyrrolo[3,4-c]carbazoles, and oxindoles (see P. S. Sharma, R. Sharma, R. Tyagi, Curr. Cancer Drug Targets 8 (2008) 53-75). WO 03/062236 identifies a series of 2-(pyridin-2-ylamino-pyrido[2,3]pyrimidin-7-ones for the treatment of Rb positive cancers that show selectivity for CDK4/6, including 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylammino)-8H-pyrido-[2,3-d]-pyrimidin-7-one (palbociclib). The clinical trial studies have reported rates of Grade 3/4 neutropenia and leukopenia with the use of palbociclib, resulting in 71% of patients requiring a dose interruption and 35% requiring a dose reduction; and adverse events leading to 10% of the discontinuations (see Finn, Abstract S1-6, SABCS 2012). These side effects may be caused by the undesirable pharmacokinetics of palbociclib, which has a relatively long T_(1/2) of roughly 26.7 hours, resulting in an accumulative concentration build-up of the CDK4/6 inhibitor and a persistent quiescence of HPSC replication.

VanderWel et al. describe an iodine-containing pyrido[2,3-d]pyrimidine-7-one (CKIA) as a potent and selective CDK4 inhibitor (see VanderWel et al., J. Med. Chem. 48 (2005) 2371-2387).

WO 99/15500 filed by Glaxo Group Ltd discloses protein kinase and serine/threonine kinase inhibitors.

WO 2010/020675 filed by Novartis AG describes pyrrolopyrimidine compounds as CDK inhibitors. WO 2011/101409 also filed by Novartis describes pyrrolopyrimidines with CDK 4/6 inhibitory activity.

WO 2005/052147 filed by Novartis and WO 2006/074985 filed by Janssen Pharma disclose additional CDK4 inhibitors.

US 2007/0179118 filed by Barvian et al. teaches the use of CDK4 inhibitors to treat inflammation.

U.S. Patent Publication 2011/0224227 to Sharpless et al. describes the use of certain CDK4/6 inhibitors, such as PD0332991 and 2BrIC (see Zhu, et al., J. Med. Chem., 46 (11) 2027-2030 (2003); PCT/US2009/059281) to reduce or prevent the effects of cytotoxic compounds on HSPCs in a subject undergoing chemotherapeutic treatments. See also U.S. Patent Publication 2012/0100100.

U.S. Patent Publication 2011/0224221 to Sharpless et al. describes the use of certain CDK4/6 inhibitors, such as PD0332991 and 2BrIC (see Zhu, et al., J. Med. Chem., 46 (11) 2027-2030 (2003); PCT/US2009/059281) to reduce or prevent the deleterious effects of ionizing radiation on HSPCs in a subject exposed to radiation. See also U.S. Patent Publication 2012/0100100.

Stone, et al., Cancer Research 56, 3199-3202 (Jul. 1, 1996) describes reversible, p16-mediated cell cycle arrest as protection from chemotherapy.

WO 2012/061156 filed by Tavares and assigned to G1 Therapeutics describes CDK inhibitors (see also, U.S. Pat. Nos. 8,829,012, 8,822,683, 8,598,186, 8,691,830, and 8,598,197, all assigned to G1 Therapeutics), describe CDK Inhibitors having the basic core structure:

WO 2013/148748 filed by Tavares and assigned to G1 Therapeutics describes Lactam Kinase inhibitors having the basic core structures:

U.S. Patent Publication 2014/0275066 and 2014/0275067, assigned to G1 Therapeutics, describes the use of CDK4/6 inhibitors such as 2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-one for the protection of healthy hematopoietic stem and progenitor cells in a subject receiving a DNA-damaging chemotherapeutic agent for the treatment of a Rb-negative tumors.

U.S. Patent Publication 2014/0274896, assigned to G1 Therapeutics, describes the use of CDK4/6 inhibitors such as 2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-one for the protection of healthy hematopoietic stem and progenitor cells in a subject exposed to ionizing radiation.

U.S. Patent Publication 2014/0271466, assigned to G1 Therapeutics, describes the use of CDK4/6 inhibitors such as 2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-one for use as an anti-neoplastic for the treatment of a Rb-positive proliferative disorders.

U.S. Patent Publication 2014/0271460, assigned to G1 Therapeutics, describes the use of CDK4/6 inhibitors such as 2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-one for use an anti-neoplastic for the treatment of a T- or B-cell disorder, for example a leukemia.

While selective CDK4/6 inhibitors are generally designed to target CDK4/6-replication dependent cancers, the very fact that they inhibit CDK4/6 activity may also result in deleterious effects to CDK4/6-dependent healthy cells, for example their growth inhibition. CDK4/6 activity is necessary for the production of healthy blood cells by the bone marrow, as healthy hematopoietic stem and progenitor cells (HSPCs) require the activity of CDK4/6 for proliferation (see Roberts et al. Multiple Roles of Cyclin-Dependent Kinase 4/6 Inhibitors in Cancer Therapy. JNCI 2012; 104(6):476-487). Healthy hematopoietic stem cells give rise to progenitor cells which in turn give rise to all the differentiated components of blood as shown in FIG. 1 (e.g., lymphocytes, erythrocytes, platelets, granulocytes, monocytes). Healthy hematopoietic cells display a gradient dependency on CDK4/6 activity for proliferation during myeloid/erythroid differentiation (see Johnson et al. Mitigation of hematological radiation toxicity in mice through pharmacological quiescence induced by CDK4/6 inhibition. J Clin. Invest. 2010; 120(7): 2528-2536). Accordingly, the least differentiated cells (e.g., healthy hematopoietic stem cells (HSCs), multi-potent progenitors (MPPs), and common myeloid progenitors (CMP)) appear to be the most dependent on CDK4/6 activity for proliferation, and therefore the most deleteriously affected by the use of a CDK4/6 inhibitor to treat a CDK4/6 replication dependent cancer or other proliferative disorder.

Accordingly, there is an ongoing need for CDK4/6 inhibitor compounds, methods, and regimes to treat patients with select Rb-positive cancers and abnormal cellular proliferative disorders while minimizing the treatment's effect on healthy cells such as HSPCs.

SUMMARY OF THE INVENTION

Tricyclic lactam compounds, methods, and compositions are provided to treat select Rb-positive abnormal cellular proliferation including an Rb-positive cancer while minimizing the treatment's deleterious effects on healthy cells, such as healthy HSPCs and other CDK4/6-replication dependent healthy cells by administration of an effective amount of a compound described herein.

In one embodiment of the invention, a compound is selected from the compounds of Formula I, II, III, IV, V, or VI as described herein, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one non-limiting example, a compound can be selected from the compounds of Table 1 below, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.

In one embodiment, the Rb-positive cancer can be Rb-positive adenocarcinoma. The Rb-positive cancer can be Rb-positive adenocarcinoma of the colon. The Rb-positive cancer can also be Rb-positive adenocarcinoma of the rectum.

Alternatively, the Rb-positive cancer can be Rb-positive anaplastic astrocytoma. The Rb-positive cancer can be Rb-positive breast cancer. In one embodiment, the Rb-positive cancer is Rb-positive estrogen-receptor positive, HER2-negative advanced breast cancer. Alternatively, the Rb-positive cancer can be Rb-positive estrogen receptor-negative breast cancer. The Rb-positive cancer can be Rb-positive estrogen receptor positive breast cancer. The Rb-positive cancer can be Rb-positive late-line metastatic breast cancer. The Rb-positive cancer can be Rb-positive luminal A breast cancer. The Rb-positive cancer can be Rb-positive luminal B breast cancer. The Rb-positive cancer can be Rb-positive Her2-negative breast cancer or Rb-positive HER2-positive breast cancer. The Rb-positive cancer is Rb-positive male breast cancer. In one embodiment, the Rb-positive cancer is Rb-positive progesterone receptor-negative breast cancer. The Rb-positive cancer can be Rb-positive progesterone receptor-positive breast cancer. The Rb-positive cancer can be Rb-positive recurrent breast cancer. In one embodiment, the Rb-positive cancer is Rb-positive stage IV breast cancers. In one embodiment, the Rb-positive cancer is Rb-positive advanced HER2-positive breast cancer.

The Rb-positive cancer can be Rb-positive bronchial cancer. The Rb-positive cancer can be Rb-positive colon cancer. The Rb-positive cancer can be Rb-positive recurrent colon cancer. The Rb-positive cancer can be Rb-positive stage IV colon cancers. In one embodiment, the Rb-positive cancer is Rb-positive colorectal cancer.

In one embodiment, the Rb-positive cancer is Rb-positive endometrial cancer.

The Rb-positive cancer can be Rb-positive extragonadal seminoma. The Rb-positive cancer can be Rb-positive stage III extragonadal seminoma. The Rb-positive cancer can be Rb-positive stage IV extragonadal seminoma.

The Rb-positive cancer can be Rb-positive germ cell cancer. The Rb-positive cancer can be Rb-positive central nervous system germ cell tumor. The Rb-positive cancer can be Rb-positive familial testicular germ cell tumor. The Rb-positive cancer can be Rb-positive recurrent gonadal germ cell tumor. The Rb-positive cancer can be Rb-positive recurrent extragonadal non-seminomatous germ cell tumor. The Rb-positive cancer can be Rb-positive extragonadal seminomatous germ cell tumor. The Rb-positive cancer can be Rb-positive recurrent malignant testicular germ cell tumors. The Rb-positive cancer can be Rb-positive recurrent ovarian germ cell tumors. The Rb-positive cancer can be Rb-positive stage III malignant testicular germ cell tumors. The Rb-positive cancer can be Rb-positive stage III ovarian germ cell tumors. The Rb-positive cancer can be Rb-positive stage IV ovarian germ cell tumors. The Rb-positive cancer can be Rb-positive stage III extragonadal non-seminomatous germ cell tumors. The Rb-positive cancer can be Rb-positive stage IV extragonadal non-seminomatous germ cell tumors. In one embodiment, the Rb-positive cancer is Rb-positive germ cell cancer. In one embodiment, the Rb-positive cancer is Rb-positive cisplatin-refractory, unresectable germ cell cancer.

In one embodiment, the Rb-positive cancer is Rb-positive glioblastoma.

In one embodiment, the Rb-positive cancer is Rb-positive liver cancer. The Rb-positive cancer can be Rb-positive hepatocellular cancer.

The Rb-positive cancer can be Rb-positive lung cancer. In one embodiment, the Rb-positive cancer is Rb-positive non-small cell lung cancer. In one embodiment, the Rb-positive cancer is Rb-positive KRAS mutant non-small cell lung cancer.

The Rb-positive cancer can be Rb-positive melanoma. In one embodiment, the Rb-positive cancer is Rb-positive recurrent melanomas. In one embodiment, the Rb-positive cancer is Rb-positive stage IV melanomas.

The Rb-positive cancer can be Rb-positive ovarian cancer. In one embodiment, the Rb-positive cancer is Rb-positive ovarian epithelial carcinoma.

The Rb-positive cancer can be Rb-positive pancreatic cancer.

The Rb-positive cancer can be Rb-positive prostate cancer.

In one embodiment, the Rb-positive cancer is Rb-positive rectal cancer. The Rb-positive cancer can be Rb-positive recurrent rectal cancer. The Rb-positive cancer can be Rb-positive stage IV rectal cancers.

The Rb-positive cancer can be Rb-positive sarcoma. The Rb-positive cancer can be Rb-positive gliosarcoma. The Rb-positive cancer can be Rb-positive liposarcoma. The Rb-positive cancer can be Rb-positive fibrosarcoma. The Rb-positive cancer can be Rb-positive myxosarcoma. In one embodiment, the Rb-positive cancer can be Rb-positive chondrosarcoma. The Rb-positive cancer can be Rb-positive osteosarcoma.

The Rb-positive cancer can be Rb-positive malignant fibrous histiocytoma. The Rb-positive cancer can be Rb-positive hemangiosarcoma. The Rb-positive cancer can be Rb-positive angiosarcoma. The Rb-positive cancer can be Rb-positive lymphangiosarcoma. The Rb-positive cancer can be Rb-positive mesothelioma. The Rb-positive cancer can be Rb-positive leiomyosarcoma. The Rb-positive cancer can be Rb-positive rhabdomyosarcoma. The Rb-positive cancer can be Rb-positive meningioma. The Rb-positive cancer can be Rb-positive schwannoma.

In one embodiment, the Rb-positive cancer is Rb-positive pheochromocytoma. The Rb-positive cancer can be Rb-positive Islet cell carcinoma. The Rb-positive cancer can be Rb-positive carcinoid. The Rb-positive cancer can be Rb-positive paraganglioma.

In one embodiment, the Rb-positive cancer is Rb-positive squamous cell carcinoma. The Rb-positive cancer can be Rb-positive adenocarcinoma. The Rb-positive cancer can be Rb-positive hepatocellular carcinoma. The Rb-positive cancer can be Rb-positive renal cell carcinoma. The Rb-positive cancer can be Rb-positive cholangiocarcinoma.

The Rb-positive cancer can be Rb-positive refractory solid tumors.

The Rb-positive cancer can be Rb-positive neuroblastoma.

The Rb-positive cancer can be Rb-positive medulloblastoma.

In one embodiment, the Rb-positive cancer is a Teratoma. The Rb-positive cancer can be Rb-positive ovarian immature teratoma. The Rb-positive cancer can be Rb-positive ovarian mature teratoma. The Rb-positive cancer can be an Rb-positive ovarian specialized teratoma. The Rb-positive cancer can be Rb-positive testicular immature teratoma. The Rb-positive cancer can be Rb-positive testicular mature teratoma. The Rb-positive cancer can be Rb-positive teratoma. The Rb-positive cancer can be Rb-positive ovarian monodermal teratoma.

The Rb-positive cancer can be Rb-positive testicular cancer.

In one embodiment, the Rb-positive cancer is Rb-positive vaginal cancer.

In one embodiment, the Rb-positive cancer is selected from an Rb-positive carcinoma, sarcoma, including, but not limited to, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, or a combination of one or more of the foregoing cancers.

In one embodiment, the subject is suffering from an Rb-positive abnormal cellular proliferation disorder. In one embodiment, the Rb-positive abnormal cellular proliferation disorder is non-cancerous.

In one embodiment, a compound described herein, when used to treat a select Rb-positive cellular proliferation disorder, such as a cancer, allows for a rapid reentry of healthy cells into the normal cell-cycle and a fast reconstitution of damaged tissue and progeny cells such as hematological cells. Non-limiting examples of active compounds are described in Table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof as provided below.

In one embodiment of the invention, a compound described herein can be administered in a concerted regimen with another agent such as a non-DNA-damaging, targeted anti-neoplastic agent or a hematopoietic growth factor agent for beneficial, additive, or synergistic effect against the abnormal cellular proliferation. It has been recently reported that the untimely administration of hematopoietic growth factors can have serious side effects. For example, the use of the EPO family of growth factors has been associated with arterial hypertension, cerebral convulsions, hypertensive encephalopathy, thromboembolism, iron deficiency, influenza like syndromes and venous thrombosis. The G-CSF family of growth factors has been associated with spleen enlargement and rupture, respiratory distress syndrome, allergic reactions and sickle cell complications. By combining the administration of a compound described herein and methods of the present invention with the timely administration of hematopoietic growth factors, for example, at the time point wherein the affected cells are no longer under growth arrest, it is possible for the health care practitioner to decrease the amount of the growth factor to minimize the unwanted adverse effects while achieving the desired therapeutic benefit. In one embodiment, the growth factor is administered upon cessation of the effect of the inhibitory effect of the compound on the CDK4/6 replication dependent healthy cells, for example HSPCs.

In one embodiment, the use of a compound or method described herein is combined with the use of a hematopoietic growth factor including, but not limited to, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), thrombopoietin, interleukin (IL)-12, steel factor, and erythropoietin (EPO), or a derivative thereof. In one embodiment, the tricylic lactam is administered prior to administration of the hematopoietic growth factor. In one embodiment, the hematopoietic growth factor administration is timed so that the tricyclic lactam's effect on HSPCs has dissipated.

In one embodiment, the use of a compound described herein is combined in a therapeutic regime with at least one other chemotherapeutic agent, and can be one that does, or in certain embodiments does not, rely on proliferation or advancement through the cell-cycle for anti-proliferative activity. Such agent may include, but is not limited to, tamoxifen, midazolam, letrozole, bortezomib, anastrozole, goserelin, an mTOR inhibitor, a PI3 kinase inhibitors, dual mTOR-PI3K inhibitors, MEK inhibitors, RAS inhibitors, ALK inhibitors, HSP inhibitors (for example, HSP70 and HSP 90 inhibitors, or a combination thereof). Examples of mTOR inhibitors include but are not limited to rapamycin and its analogs, everolimus (Afinitor), temsirolimus, ridaforolimus, sirolimus, and deforolimus. Examples of P13 kinase inhibitors include but are not limited to Wortmannin, demethoxyviridin, perifosine, idelalisib, PX-866, IPI-145, BAY 80-6946, BEZ235, RP6503, TGR 1202 (RP5264), MLN1117 (INK1117), Pictilisib, Buparlisib, SAR245408 (XL147), SAR245409 (XL765), Palomid 529, ZSTK474, PWT33597, RP6530, CUDC-907, and AEZS-136. Examples of MEK inhibitors include but are not limited to Tametinib, Selumetinib, MEK162, GDC-0973 (XL518), and PD0325901. Examples of RAS inhibitors include but are not limited to Reolysin and siGl2D LODER. Examples of ALK inhibitors include but are not limited to Crizotinib, AP26113, and LDK378. HSP inhibitors include but are not limited to Geldanamycin or 17-N-Allylamino-17-demethoxygeldanamycin (17AAG), and Radicicol.

In certain embodiments, a compound described herein is administered to the subject prior to treatment with another chemotherapeutic agent, during treatment with another chemotherapeutic agent, after administration of another chemotherapeutic agent, or a combination thereof. In one embodiment, a compound described herein is administered to the subject less than about 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, or ½ hour or less prior to treatment with another chemotherapeutic agent in order to sensitize the Rb-positive cancer to the chemotherapeutic agent's effects.

In one embodiment, a compound described herein is administered in a manner that allows the drug facile access to the blood stream, for example via intravenous injection or sublingual, intraaortal, or other efficient blood-stream accessing route. In one embodiment, a compound described herein is administered in an orally administrable formulation. In other embodiments, a compound described herein is administered via topical, transdermal, or other desired administrative routes.

In one embodiment, a compound described herein is administered to the subject less than about 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, or ½ hour or less prior to treatment with the hematopoietic growth factor. In one embodiment, the compound is administered up to 4 hours prior to treatment with the hematopoietic growth factor or other chemotherapeutic agent.

The use of a compound as described herein in a therapeutic regime targeting CDK4/6-replication dependent cancers can result in reduced anemia, reduced lymphopenia, reduced thrombocytopenia, or reduced neutropenia compared to that typically expected after, common after, or associated with treatment with currently available antineoplastic chemotherapeutic agents. The use of the compounds as described herein may result in a faster recovery from bone marrow suppression associated with long-term use of CDK4/6 inhibitors, such as myelosuppression, anemia, lymphopenia, thrombocytopenia, or neutropenia, following the cessation of use of the CDK4/6 inhibitor. In some embodiments, the use of a compound as described herein results in reduced bone marrow suppression associated with long-term use of CDK4/6 inhibitors, such as myelosuppression, anemia, lymphopenia, leukopenia, thrombocytopenia, or granulocytopenias such as neutropenia.

In some embodiments, the subject or host is a mammal, including a human. The compound can be administered to the subject by any desired route, including intravenous, sublingual, buccal, oral, intraaortal, topical, intranasal, parenteral, transdermal, systemic, intramuscular, or via inhalation.

In summary, the present invention includes the following features:

-   A) Tricyclic lactam compounds, methods, and compositions as     chemotherapeutics which minimize the deleterious effects on CDK4/6     replication dependent healthy cells, for example hematopoietic stem     and progenitor cells (HSPCs), in a subject undergoing treatment for     a select Rb-positive cancer, comprising administering an effective     amount of a tricyclic lactam compound of Formula I, II, III, IV, V,     or VI, including a compound selected from Table 1 as described     herein; -   B) Tricyclic lactam compounds, methods, and compositions as     chemotherapeutics which minimize the deleterious effects on CDK4/6     replication dependent healthy cells in a subject, the method     comprising administering to a subject with an Rb-positive abnormal     cellular proliferative disorder an effective amount of a tricyclic     lactam compound of Formula I, II, III, IV, V, or VI, including a     compound selected from Table 1 as described herein. -   C) A compound as described herein, or a pharmaceutically acceptable     composition, salt, isotopic analog, or prodrug thereof, for use as a     chemotherapeutic in the treatment of an Rb-positive abnormal     cellular proliferation disorder, including Rb-positive cancers; -   D) A compound as described herein, or a pharmaceutically acceptable     composition, salt, isotopic analog, and prodrug thereof, for use as     a chemotherapeutic regimen for the treatment of an Rb-positive     abnormal cellular proliferation disorder, including Rb-positive     cancers, which minimizes the deleterious effects on     CDK4/6-replication dependent healthy cells, for example HSPCs or     renal cells; -   E) A compound as described herein, or a pharmaceutically acceptable     composition, salt, isotopic analog, and prodrug thereof, for use in     combination with hematopoietic growth factors in a subject     undergoing a therapeutic regime to treat an Rb-positive abnormal     cellular proliferation disorder, including Rb-positive cancers; -   F) Compounds as described herein, or a pharmaceutically acceptable     composition, salt, isotopic analog, or prodrug thereof, for use in     combination with a second chemotherapeutic agent in a subject     undergoing a therapeutic regime to treat an Rb-positive abnormal     cellular proliferation disorder, including Rb-positive cancers; -   G) Use of a compound described herein, or a pharmaceutically     acceptable composition, salt, isotopic analog, or prodrug thereof,     in the manufacture of a medicament for use as a chemotherapeutic to     treat a subject with an Rb-positive abnormal cellular proliferation     disorder, including Rb-positive cancers; -   H) Use of a compound described herein, or a pharmaceutically     acceptable composition, salts, isotopic analog, or prodrug thereof,     in the manufacture of a medicament for use as chemotherapeutic to     treat a subject with an Rb-positive cellular proliferation disorder,     including a Rb-positive cancer that, when exposed to a CDK4/6     inhibitor, is growth arrested or growth inhibited; -   I) Processes for the preparation of therapeutic products that     contain an effective amount of a compound described herein, for use     in the treatment of a subject having an Rb-positive abnormal     cellular proliferation disorder, such as cancer, and; -   J) A method for manufacturing a medicament selected from the     compounds described herein intended for therapeutic use as a     chemotherapeutic on the treatment of an Rb-positive, abnormal     cellular proliferation disorder, such as a cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of hematopoiesis showing the hierarchical proliferation of healthy hematopoietic stem cells (HSC) and healthy hematopoietic progenitor cells with increasing differentiation upon proliferation.

FIGS. 2-4 illustrate several exemplary embodiments of R² of the compounds of the invention.

FIGS. 5A-5C, 6A-6D, 7A-7C, 8A-8B, and 9A-9F illustrate several exemplary embodiments of the core structure of the compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Tricyclic lactam compounds, methods, and compositions are provided as chemotherapeutics for the treatment of select Rb-positive cancers which minimize or reduce the deleterious effects on CDK4/6 replication dependent healthy cells, such as hematopoietic stem cells and/or progenitor cells (HSPCs) in subjects, typically humans.

Definitions

Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Definition of standard chemistry terms may be found in reference works, including Carey and Sundberg (2007) Advanced Organic Chemistry 5^(th) Ed. Vols. A and B, Springer Science+Business Media LLC, New York. The practice of the present invention will employ, unless otherwise indicated, conventional methods of synthetic organic chemistry, mass spectroscopy, preparative and analytical methods of chromatography, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology. Conventional methods of organic chemistry include those included in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6^(th) Edition, M. B. Smith and J. March, John Wiley & Sons, Inc., Hoboken, N.J., 2007.

The term “alkyl,” either alone or within other terms such as “haloalkyl” and “alkylamino,” embraces linear or branched radicals having one to about twelve carbon atoms. “Lower alkyl” radicals have one to about six carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, hexyl and the like. The term “alkylene” embraces bridging divalent linear and branched alkyl radicals. Examples include methylene, ethylene, propylene, isopropylene and the like.

The term “alkenyl” embraces linear or branched radicals having at least one carbon-carbon double bond of two to about twelve carbon atoms. “Lower alkenyl” radicals having two to about six carbon atoms. Examples of alkenyl radicals include ethenyl, propenyl, allyl, propenyl, butenyl and 4-methylbutenyl. The terms “alkenyl” and “lower alkenyl,” embrace radicals having “cis” and “trans” orientations, or alternatively, “E” and “Z” orientations.

The term “alkynyl” denotes linear or branched radicals having at least one carbon-carbon triple bond and having two to about twelve carbon atoms. “Lower alkynyl” radicals having two to about six carbon atoms. Examples of such radicals include propargyl, butynyl, and the like.

Alkyl, alkenyl, and alkynyl radicals may be optionally substituted with one or more functional groups such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.

The term “alkylamino” embraces “N-alkylamino” and “N,N-dialkylamino” where amino groups are independently substituted with one alkyl radical and with two alkyl radicals, respectively. “Lower alkylamino” radicals have one or two alkyl radicals of one to six carbon atoms attached to a nitrogen atom. Suitable alkylamino radicals may be mono or dialkylamino such as N-methylamino, N-ethylamino, N.N-dimethylamino, N,N-diethylamino and the like.

The term “halo” means halogens such as fluorine, chlorine, bromine or iodine atoms.

The term “haloalkyl” embraces radicals wherein any one or more of the alkyl carbon atoms is substituted with one or more halo as defined above. Examples include monohaloalkyl, dihaloalkyl and polyhaloalkyl radicals including perhaloalkyl. A monohaloalkyl radical, for one example, may have an iodo, bromo, chloro or fluoro atom within the radical. Dihalo and polyhaloalkyl radicals may have two or more of the same halo atoms or a combination of different halo radicals. “Lower haloalkyl” embraces radicals having 1-6 carbon atoms. Examples of haloalkyl radicals include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. “Perfluoroalkyl” means an alkyl radical having all hydrogen atoms replaced with fluoro atoms. Examples include trifluoromethyl and pentafluoroethyl.

The term “aryl”, alone or in combination, means a carbocyclic aromatic system containing one or two rings wherein such rings may be attached together in a fused manner. The term “aryl” embraces aromatic radicals such as phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl. More preferred aryl is phenyl. Said “aryl” group may have 1 or more substituents such as lower alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy, lower alkylamino, and the like. An aryl group may be optionally substituted with one or more functional groups such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.

The term “heterocyclyl” (or “heterocyclo”) embraces saturated, and partially saturated heteroatom-containing ring radicals, where the heteroatoms may be selected from nitrogen, sulfur and oxygen. Heterocyclic rings comprise monocyclic 6-8 membered rings, as well as 5-16 membered bicyclic ring systems (which can include bridged fused and spiro-fused bicyclic ring systems). It does not include rings containing —O—O—.—O—S— or —S—S— portions. Said “heterocyclyl” group may have 1 to 3 substituents such as hydroxyl, Boc, halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, lower alkoxy, amino, lower alkylamino, and the like.

Examples of saturated heterocyclo groups include saturated 3- to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms [e.g. pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl, piperazinyl]; saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g. morpholinyl]; saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g., thiazolidinyl]. Examples of partially saturated heterocyclyl radicals include dihydrothienyl, dihydropyranyl, dihydrofuryl, dihydrothiazolyl, and the like.

Particular examples of partially saturated and saturated heterocyclo groups include pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl, pyrazolidinyl, piperazinyl, morpholinyl, tetrahydropyranyl, thiazolidinyl, dihydrothienyl, 2,3-dihydro-benzo[1,4]dioxanyl, indolinyl, isoindolinyl, dihydrobenzothienyl, dihydrobenzofuryl, isochromanyl, chromanyl, 1,2-dihydroquinolyl, 1,2,3,4-tetrahydro-isoquinolyl, 1,2,3,4-tetrahydro-quinolyl, 2,3,4,4a,9,9a-hexahydro-1H-3-aza-fluorenyl, 5,6,7-trihydro-1,2,4-triazolo[3,4-a]isoquinolyl, 3,4-dihydro-2H-benzo[1,4]oxazinyl, benzo[1,4]dioxanyl, 2,3-dihydro-1H-1λ′-benzo [d]isothiazol-6-yl, dihydropyranyl, dihydrofuryl and dihydrothiazolyl, and the like.

Heterocyclo groups also includes radicals where heterocyclic radicals are fused/condensed with aryl radicals: unsaturated condensed heterocyclic group containing 1 to 5 nitrogen atoms, for example, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl [e.g., tetrazolo[1,5-b]pyridazinyl]; unsaturated condensed heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g. benzoxazolyl, benzoxadiazolyl]; unsaturated condensed heterocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g., benzothiazolyl, benzothiadiazolyl]; and saturated, partially unsaturated and unsaturated condensed heterocyclic group containing 1 to 2 oxygen or sulfur atoms [e.g. benzofuryl, benzothienyl, 2,3-dihydro-benzo[1,4]dioxinyl and dihydrobenzofuryl].

The term “heteroaryl” denotes aryl ring systems that contain one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized. Examples include unsaturated 5 to 6 membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, for example, pyrrolyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl [e.g., 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl]; unsaturated 5- to 6-membered heteromonocyclic group containing an oxygen atom, for example, pyranyl, 2-furyl, 3-furyl, etc.; unsaturated 5 to 6-membered heteromonocyclic group containing a sulfur atom, for example, 2-thienyl, 3-thienyl, etc.; unsaturated 5- to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl [e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl]; unsaturated 5 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, for example, thiazolyl, thiadiazolyl [e.g., 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl].

The term “heteroarylalkyl” denotes alkyl radicals substituted with a heteroaryl group. Examples include pyridylmethyl and thienylethyl.

The term “sulfonyl”, whether used alone or linked to other terms such as alkylsulfonyl, denotes respectively divalent radicals —SO₂—.

The terms “carboxy” or “carboxyl”, whether used alone or with other terms, such as “carboxyalkyl”, denotes —C(O)—OH.

The term “carbonyl”, whether used alone or with other terms, such as “aminocarbonyl”, denotes —C(O)—.

The term “aminocarbonyl” denotes an amide group of the Formula —C(O)—NH₂.

The terms “heterocycloalkyl” embrace heterocyclic-substituted alkyl radicals. Examples include piperidylmethyl and morpholinylethyl.

The term “arylalkyl” embraces aryl-substituted alkyl radicals. Examples include benzyl, diphenylmethyl and phenylethyl. The aryl in said aralkyl may be additionally substituted with halo, alkyl, alkoxy, halkoalkyl and haloalkoxy.

The term “cycloalkyl” includes saturated carbocyclic groups of 3 to 10 carbons. Lower cycloalkyl groups include C₃-C₆ rings. Examples include cyclopentyl, cyclopropyl, and cyclohexyl. Cycloalkyl groups may be optionally substituted with one or more functional groups such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like. The term “cycloalkylalkyl” embraces cycloalkyl-substituted alkyl radicals. “Lower cycloalkylalkyl” radicals are cycloalkyl radicals attached to alkyl radicals having one to six carbon atoms. Examples of include cyclohexylmethyl. The cycloalkyl in said radicals may be additionally substituted with halo, alkyl, alkoxy and hydroxy.

The term “cycloalkenyl” includes carbocyclic groups having one or more carbon-carbon double bonds including “cycloalkyldienyl” compounds. Examples include cyclopentenyl, cyclopentadienyl, cyclohexenyl and cycloheptadienyl.

The term “comprising” is meant to be open ended, including the indicated component but not excluding other elements.

The term “oxo” as used herein contemplates an oxygen atom attached with a double bond.

The term “nitro” as used herein contemplates —NO₂.

The term “cyano” as used herein contemplates —CN.

As used herein, the term “prodrug” means a compound which when administered to a host in vivo is converted into the parent drug. As used herein, the term “parent drug” means any of the presently described chemical compounds that are useful to treat any of the disorders described herein, or to control or improve the underlying cause or symptoms associated with any physiological or pathological disorder described herein in a host, typically a human. Prodrugs can be used to achieve any desired effect, including to enhance properties of the parent drug or to improve the pharmaceutic or pharmacokinetic properties of the parent. Prodrug strategies exist which provide choices in modulating the conditions for in vivo generation of the parent drug, all of which are deemed included herein. Nonlimiting examples of prodrug strategies include covalent attachment of removable groups, or removable portions of groups, for example, but not limited to acylation, phosphorylation, phosphonylation, phosphoramidate derivatives, amidation, reduction, oxidation, esterification, alkylation, other carboxy derivatives, sulfoxy or sulfone derivatives, carbonylation or anhydride, among others.

Throughout the specification and claims, a given chemical formula or name shall encompass all optical and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist, unless otherwise noted.

The current invention is directed to an HSPC-sparing strategy during the treatment of Rb-positive proliferation disorders. According, as used herein, the term “HSPCs” is meant to describe healthy hematopoietic stem and/or hematopoietic progenitor cells, as opposed to diseased HSPCs or cells of related hematological origin. HSPCs include hematopoietic stem cells, such as long term hematopoietic stem cells (LT-HSCs) and short term hematopoietic stem cells (ST-HSCs), and hematopoietic progenitor cells, including multipotent progenitors (MPPs), common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythroid progenitors (MEPs).

In some embodiments, a CDK4/6-replication dependent healthy cell is a hematopoietic stem progenitor cell. In some embodiments, the CDK4/6-replication dependent healthy cell may be a cell in a non-hematopoietic tissue, such as, but not limited to, the liver, kidney, pancreas, brain, lung, adrenals, intestine, gut, stomach, skin, auditory system, bone, bladder, ovaries, uterus, testicles, gallbladder, thyroid, heart, pancreatic islets, blood vessels, and the like.

The term “selective CDK4/6 inhibitor” used in the context of the compounds described herein includes compounds that inhibit CDK4 activity, CDK6 activity, or both CDK4 and CDK6 activity at an IC₅₀ molar concentration at least about 500, or 1000, or 1500, or 1800, or 2000, or 5000, or 10,000 times less than the IC₅₀ molar concentration necessary to inhibit to the same degree of CDK2 activity in a standard phosphorylation assay.

As used herein the term “chemotherapy” or “chemotherapeutic agent” refers to treatment with a cytostatic or cytotoxic agent (i.e., a compound) to reduce or eliminate the growth or proliferation of undesirable cells, for example cancer cells. Thus, as used herein, “chemotherapy” or “chemotherapeutic agent” refers to a cytotoxic or cytostatic agent used to treat a proliferative disorder, for example cancer.

By “induces G1-arrest” is meant that the inhibitor compound induces a quiescent state in a substantial portion of a cell population at the G1 phase of the cell cycle.

By “hematological deficiency” is meant reduced hematological cell lineage counts or the insufficient production of blood cells (i.e., myelodysplasia) and/or lymphocytes (i.e., lymphopenia, the reduction in the number of circulating lymphocytes, such as B- and T-cells).

Hematological deficiency can be observed, for example, as myelosuppression in form of anemia, reduction in platelet count (i.e., thrombocytopenia), reduction in white blood cell count (i.e., leukopenia), or the reduction in granulocytes (e.g., neutropenia).

By “synchronous reentry into the cell cycle” is meant that CDK4/6-replication dependent healthy cells, for example HSPCs, in G1-arrest due to the effect of a tricyclic lactam compound reenter the cell-cycle within relatively the same collective timeframe or at relatively the same rate upon dissipation of the compound's effect. Comparatively, by “asynchronous reentry into the cell cycle” is meant that the healthy cells, for example HSPCs, in G1 arrest reenter the cell-cycle within relatively different collective timeframes or at relatively different rates upon dissipation of the compound's effect, such as PD0332991.

By “off-cycle” or “drug holiday” is meant a time period during which the subject is not administered or exposed to a chemotherapeutic. For example, in a treatment regime wherein the subject is administered the chemotherapeutic for 21 straight days and is not administered the chemotherapeutic for 7 days, and the regime is repeated a number of times, the 7 day period of non-administration is considered the “off-cycle” or “drug holiday.” Off-cycle and drug holiday may also refer to an interruption in a treatment regime wherein the subject is not administered the chemotherapeutic for a time due to a deleterious side effect, for example, myelosuppression.

The subject treated is typically a human subject, although it is to be understood the methods described herein are effective with respect to other animals, such as mammals and vertebrate species. More particularly, the term subject can include animals used in assays such as those used in preclinical testing including but not limited to mice, rats, monkeys, dogs, pigs and rabbits; as well as domesticated swine (pigs and hogs), ruminants, equine, poultry, felines, bovines, murines, canines, and the like.

Active Compounds

In one embodiment, the invention is directed to compounds or the use of such compounds of Formula I, II, III, IV, or V:

or a pharmaceutically acceptable salt thereof;

-   wherein: -   Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)— wherein     z is 2, 3 or 4; -   each X is independently CH or N; -   each X′ is independently CH or N; -   X″ is independently CH₂, S or NH, arranged such that the moiety is a     stable 5-membered ring; R, R⁸, and R¹¹ are independently H, C₁-C₃     alkyl or haloalkyl, cycloalkyl or cycloalkyl containing one or more     heteroatoms selected from N, O or S; -(alkylene)_(m)-C₃-C₈     cycloalkyl, -(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,     -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,     -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,     -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any     of which may be optionally independently substituted with one or     more R^(x) groups as allowed by valence, and wherein two R^(x)     groups bound to the same or adjacent atoms may optionally combine to     form a ring; -   each R¹ is independently aryl, alkyl, cycloalkyl or haloalkyl,     wherein each of said alkyl, cycloalkyl and haloalkyl groups     optionally includes O or N heteroatoms in place of a carbon in the     chain and two R¹'s on adjacent ring atoms or on the same ring atom     together with the ring atom(s) to which they are attached optionally     form a 3-8-membered cycle; -   y is 0, 1, 2, 3 or 4; -   R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,     -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴;     -(alkylene)_(m)-C(O)—O-alkyl; -(alkylene)_(m)-O—R⁵,     -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any     of which may be optionally independently substituted with one or     more R^(x) groups as allowed by valence, and wherein two R^(x)     groups bound to the same or adjacent atom may optionally combine to     form a ring and wherein m is 0, 1 or 2 and n is 0, 1 or 2; -   R³ and R⁴ at each occurrence are independently:     -   (i) hydrogen or     -   (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl,         cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl         any of which may be optionally independently substituted with         one or more R^(x) groups as allowed by valence, and wherein two         R^(x) groups bound to the same or adjacent atom may optionally         combine to form a ring; or R³ and R⁴ together with the nitrogen         atom to which they are attached may combine to form a         heterocyclo ring optionally independently substituted with one         or more R^(x) groups as allowed by valence, and wherein two         R^(x) groups bound to the same or adjacent atom may optionally         combine to form a ring; -   R⁵ and R⁵* at each occurrence is:     -   (i) hydrogen or     -   (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,         heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or         heteroarylalkyl any of which may be optionally independently         substituted with one or more R^(x) groups as allowed by valence; -   R^(x) at each occurrence is independently, halo, cyano, nitro, oxo,     alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,     heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl,     cycloalkylalkyl, heterocycloalkyl, -(alkylene)_(m)-OR⁵,     -(alkylene)_(m)-O-alkylene-OR⁵, -(alkylene)_(m)-S(O)_(n)—R⁵,     -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵,     -(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵,     -(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵,     -(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴,     -(alkylene)_(m)-C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—NR³R⁴,     -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—R⁵,     -(alkylene)_(m)-N(R³)—C(S)—R⁵, -(alkylene)_(m)-O—C(O)—NR³R⁴,     -(alkylene)_(m)-O—C(S)—NR³R⁴, -(alkylene)_(m)-SO₂—NR³R⁴,     -(alkylene)_(m)-N(R³)—SO₂—R⁵, -(alkylene)_(m)-N(R³)—SO₂—NR³R⁴,     -(alkylene)_(m)-N(R³)—C(O)—OR⁵) -(alkylene)_(m)-N(R³)—C(S)—OR⁵, or     -(alkylene)_(m)-N(R³)—SO₂—R⁵; wherein:     -   said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,         cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,         heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups         may be further independently substituted with one or more         -(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵*,         -(alkylene)_(m)-S(O)_(n)—R⁵*, -(alkylene)_(m)-NR³*R⁴*,         -(alkylene)_(m)-C(O)—R⁵*, -(alkylene)_(m)-C(═S)R⁵*,         -(alkylene)_(m)-C(═O)OR⁵*, -(alkylene)_(m)-OC(═O)R⁵*,         -(alkylene)_(m)-C(S)—OR⁵*, -(alkylene)_(m)-C(O)—NR³*R⁴*,         -(alkylene)_(m)-C(S)—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,         -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,         -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,         -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*,         -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or         -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,     -   n is 0, 1 or 2, and     -   m is 0, 1 or 2; -   R³* and R⁴* at each occurrence are independently:     -   (i) hydrogen or     -   (ii) alkyl, alkenyl, alkynyl cycloalkyl, heterocyclo, aryl,         heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or         heteroarylalkyl any of which may be optionally independently         substituted with one or more R^(x) groups as allowed by valence;         or R³* and R⁴* together with the nitrogen atom to which they are         attached may combine to form a heterocyclo ring optionally         independently substituted with one or more R^(x) groups as         allowed by valence; and -   R⁶ is H or lower alkyl, -(alkylene)m-heterocyclo,     -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,     -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,     -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any     of which may be optionally independently substituted with one or     more R^(x) groups as allowed by valence, and wherein two R^(x)     groups bound to the same or adjacent atoms may optionally combine to     form a ring; and -   R¹⁰ is (i) NHR^(A), wherein R^(A) is unsubstituted or substituted     C₁-C₈ alkyl, cycloalkylalkyl, or -TT-RR, C₁-C₈ cycloalkyl or     cycloalkyl containing one or more heteroatoms selected from N, O,     and S; TT is an unsubstituted or substituted C₁-C₈ alkyl or C₃-C₈     cycloalkyl linker; and RR is a hydroxyl, unsubstituted or     substituted C₁-C₆ alkoxy, amino, unsubstituted or substituted C₁-C₆     alkylamino, unsubstituted or substituted di-C₁-C₆ alkylamino,     unsubstituted or substituted C₆-C₁₀ aryl, unsubstituted or     substituted heteroaryl comprising one or two 5- or 6-member rings     and 1-4 heteroatoms selected from N, O and S, unsubstituted or     substituted C₃-C₁₀ carbocycle, or unsubstituted or substituted     heterocycle comprising one or two 5- or 6-member rings and 1-4     heteroatoms selected from N, O and S; or (ii) —C(O)—R¹² or     —C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³ is R^(A); -   when compounds comprise a double bond in the 6-membered ring fused     to the pyrimidine ring, two R⁸ groups are present and are as defined     above; -   when compounds do not comprise a double bond in the 6-membered ring     fused to the pyrimidine ring, four R⁸ groups are present and are as     defined above; -   or a pharmaceutically acceptable salt, prodrug or isotopic variant,     for example, partially or fully deuterated form thereof.

In one embodiment, two R⁸ groups bonded to the same carbon can form an exocyclic double bond. In another embodiment, two R⁸ groups bonded to the same carbon can form a carbonyl group.

In one embodiment, the invention is directed to compounds or the use of such compounds of Formula VI:

wherein R, R¹, R², R³, R⁴, R⁵, R⁶, R^(x), Z, m, n, and y are as defined above;

-   each R¹⁴ is independently H, C₁-C₃ alkyl (including methyl) or     haloalkyl, cycloalkyl or cycloalkyl containing one or more     heteroatoms selected from N, O or S; -(alkylene)_(m)-C₃-C₈     cycloalkyl, -(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,     -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,     -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,     -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any     of which may be optionally independently substituted with one or     more R^(x) groups as allowed by valence, and wherein two R^(x)     groups bound to the same or adjacent atoms may optionally combine to     form a ring; -   or two R¹⁴ groups bonded to the same carbon can form an exocyclic     double bond; -   or two R¹⁴ groups bonded to the same carbon can form a carbonyl     group; and -   when the compound of Formula VI has a double bond, as indicated by     the (----), in the 6-membered ring fused to the pyrimidine ring, two     R¹⁴ groups are present as allowed for in Formula VI above; or -   when the compound of Formula VI does not include a double bond, as     indicated by the (----), in the 6-membered ring fused to the     pyrimidine ring, four R¹⁴ groups are present as allowed for in     Formula VI above; -   or a pharmaceutically acceptable salt, prodrug or isotopic variant,     for example, partially or fully deuterated form thereof.

In an alternative embodiment, the invention is directed to compounds or the use of such compounds of Formula I, II, III, IV, or V:

or a pharmaceutically acceptable salt thereof;

-   wherein: -   Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)— wherein     z is 2, 3 or 4; -   each X is independently CH or N; -   each X′ is independently CH or N; -   X″ is independently CH₂, S or NH, arranged such that the moiety is a     stable 5-membered ring; R, R⁸, and R¹¹ are independently H, C₁-C₃     alkyl (including methyl) or haloalkyl, cycloalkyl or cycloalkyl     containing one or more heteroatoms selected from N, O or S;     -(alkylene)_(m)-C₃-C₈ cycloalkyl, -(alkylene)_(m)-aryl,     -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,     -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴;     -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or     -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any of which, other than heterocyclo,     may be optionally independently substituted with one or more R^(x)     groups as allowed by valence, and wherein two R^(x) groups bound to     the same or adjacent atoms may optionally combine to form a ring; -   each R¹ is independently aryl, alkyl, cycloalkyl or haloalkyl,     wherein each of said alkyl, cycloalkyl and haloalkyl groups     optionally includes O or N heteroatoms in place of a carbon in the     chain and two R¹'s on adjacent ring atoms or on the same ring atom     together with the ring atom(s) to which they are attached optionally     form a 3-8-membered cycle; -   y is 0, 1, 2, 3 or 4; -   R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,     -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴;     -(alkylene)_(m)-C(O)—O-alkyl; -(alkylene)_(m)-O—R⁵,     -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any     of which, other than heterocyclo, may be optionally independently     substituted with one or more R^(x) groups as allowed by valance, and     wherein two R^(x) groups bound to the same or adjacent atom may     optionally combine to form a ring and wherein m is 0, 1, or 2 and n     is 0, 1 or 2; -   wherein heterocyclo may be optionally independently substituted with     1 to 3 R^(x) groups as allowed by valance, and wherein two R^(x)     groups bound to the same or adjacent atom may optionally combine to     form a ring; -   R³ and R⁴ at each occurrence are independently:     -   (i) hydrogen or     -   (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl,         cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl         any of which, other than heterocyclo, may be optionally         independently substituted with one or more R^(x) groups as         allowed by valance, and wherein two R^(x) groups bound to the         same or adjacent atom may optionally combine to form a ring; or         R³ and R⁴ together with the nitrogen atom to which they are         attached may combine to form a heterocyclo ring optionally         independently substituted with one or more R^(x) groups as         allowed by valance, and wherein two R^(x) groups bound to the         same or adjacent atom may optionally combine to form a ring; -   R⁵ and R⁵* at each occurrence is:     -   (i) hydrogen or     -   (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,         heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or         heteroarylalkyl any of which, other than heterocyclo, may be         optionally independently substituted with one or more R^(x)         groups as allowed by valance; -   R^(x) at each occurrence is independently, halo, cyano, nitro, oxo,     alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,     heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl,     cycloalkylalkyl, heterocycloalkyl, -(alkylene)_(m)-OR⁵,     -(alkylene)_(m)-O-alkylene-OR⁵, -(alkylene)_(m)-S(O)_(n)—R⁵,     -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵,     -(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵,     -(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵,     -(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴,     -(alkylene)_(m)-C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—NR³R⁴,     -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—R⁵,     -(alkylene)_(m)-N(R³)—C(S)—R⁵, -(alkylene)_(m)-O—C(O)—NR³R⁴,     -(alkylene)_(m)-O—C(S)—NR³R⁴, -(alkylene)_(m)-SO₂—NR³R⁴,     -(alkylene)_(m)-N(R³)—SO₂—R⁵, -(alkylene)_(m)-N(R³)—SO₂—NR³R⁴,     -(alkylene)_(m)-N(R³)—C(O)—OR⁵, -(alkylene)_(m)-N(R³)—C(S)—OR⁵, or     -(alkylene)_(m)-N(R³)—SO₂—R⁵; wherein:     -   said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,         cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,         heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups,         any of which, other than heterocyclo, may be further         independently substituted with one or more -(alkylene)_(m)-CN,         -(alkylene)_(m)-OR⁵*, -(alkylene)_(m)-S(O)_(n)—R⁵*,         -(alkylene)_(m)-NR³*R⁴*, -(alkylene)_(m)-C(O)—R⁵*,         -(alkylene)_(m)-C(═S)R⁵*, -(alkylene)_(m)-C(═O)OR⁵*,         -(alkylene)_(m)-OC(═O)R⁵*, -(alkylene)_(m)-C(S)—OR⁵*,         -(alkylene)_(m)-C(O)—NR³*R⁴*, -(alkylene)_(m)-C(S)—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,         -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,         -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,         -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*,         -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or         -(alkylene)_(m)-N(R³*)—SO₂—R⁵*, and     -   wherein heterocycle may be further independently substituted         with one to three substitutions selected from     -   -(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵*,         -(alkylene)_(m)-S(O)_(n)—R⁵*, -(alkylene)_(m)-NR³*R⁴*,         -(alkylene)_(m)-C(O)—R⁵*, -(alkylene)_(m)-C(═S)R⁵*,         -(alkylene)_(m)-C(═O)OR⁵*, -(alkylene)_(m)-OC(═O)R⁵*,         -(alkylene)_(m)-C(S)—OR⁵*, -(alkylene)_(m)-C(O)—NR³*R⁴*,         -(alkylene)_(m)-C(S)—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,         -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,         -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,         -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,         -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*,         -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or         -(alkylene)_(m)-N(R³*)—SO₂—R⁵* ;     -   n is 0, 1 or 2, and     -   m is 0, 1; or 2 and -   R³* and R⁴* at each occurrence are independently:     -   (i) hydrogen or     -   (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,         heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or         heteroarylalkyl any of which, other than heterocyclo, may be         optionally independently substituted with one or more R^(x)         groups as allowed by valance; or R³* and R⁴* together with the         nitrogen atom to which they are attached may combine to form a         heterocyclo ring optionally independently substituted with one         or more R^(x) groups as allowed by valance; -   R⁶ is H, absent, or lower alkyl, -(alkylene)m-heterocyclo,     -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -   -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,     -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any     of which, other than heterocyclo, may be optionally independently     substituted with one or more R^(x) groups as allowed by valence, and     wherein two R^(x) groups bound to the same or adjacent atoms may     optionally combine to form a ring; and -   R¹⁰ is (i) NHR^(A), wherein R^(A) is unsubstituted or substituted     C₁-C₈ alkyl, cycloalkylalkyl, or -TT-RR, C₁-C₈ cycloalkyl or     cycloalkyl containing one or more heteroatoms selected from N, O,     and S; TT is an unsubstituted or substituted C₁-C₈ alkyl or C₃-C₈     cycloalkyl linker; and RR is a hydroxyl, unsubstituted or     substituted C₁-C₆ alkoxy, amino, unsubstituted or substituted C₁-C₆     alkylamino, unsubstituted or substituted di-C₁-C₆ alkylamino,     unsubstituted or substituted C₆-C₁₀ aryl, unsubstituted or     substituted heteroaryl comprising one or two 5- or 6-member rings     and 1-4 heteroatoms selected from N, O and S, unsubstituted or     substituted C₃-C₁₀ carbocycle, or unsubstituted or substituted     heterocycle comprising one or two 5- or 6-member rings and 1-4     heteroatoms selected from N, O and S; or (ii) —C(O)—R¹² or     —C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³ is R^(A); -   when the compound of Formula I, II, III, IV, or V has a double bond,     as indicated by the (----), in the 6-membered ring fused to the     pyrimidine ring, two R⁸ groups are present as allowed for in Formula     I, II, III, IV, or V above; or -   when the compound of Formula I, II, III, IV, or V does not include a     double bond, as indicated by the (----), in the 6-membered ring     fused to the pyrimidine ring, four R⁸ groups are present as allowed     for in Formula I, II, III, IV, or V above; -   wherein each heteroaryl is an aryl ring system that contains one or     more heteroatoms selected from the group O, N and S, wherein the     ring nitrogen and sulfur atom(s) are optionally oxidized, and     nitrogen atom(s) are optionally quarternized; -   wherein each aryl is a carbocyclic aromatic system containing one or     two rings, wherein such rings may be attached together in a fused     manner, and wherein each aryl may have 1 or more R^(x) substituents; -   wherein each heterocyclo is a saturated or partially saturated     heteroatom-containing ring radical, where the heteroatoms may be     selected from nitrogen, sulfur and oxygen, wherein each heterocyclo     is a monocyclic 6-8 membered ring or a 5-16 membered bicyclic ring     system, and wherein each heterocyclo may have 1 to 3 R^(x)     substituents;     or a pharmaceutically acceptable salt, prodrug or isotopic variant,     for example, partially or fully deuterated form thereof.

In an alternative embodiment, the term “aryl” means a carbocyclic aromatic system containing one or two rings wherein such rings may be attached together in a fused manner, which may have 1 or more substituents selected from lower alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy and lower alkylamino,

In an alternative embodiment, the term “heterocyclyl” or “heterocyclo” means a saturated or partially saturated heteroatom-containing ring radical, where the heteroatoms may be selected from nitrogen, sulfur and oxygen, which may have 1 to 3 substituents selected from hydroxyl, Boc, halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, lower alkoxy, amino and lower alkylamino, wherein the heterocyclic ring is a monocyclic 6-8 membered rings, or a 5-16 membered bicyclic ring systems which can include bridged fused and spiro-fused bicyclic ring systems, and which does not include rings containing —O—O—.—O—S— or —S—S— portion.

In an alternative embodiment, the term “heteroaryl” means an aryl ring system that contains one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized;

In one embodiment, two R⁸ groups bonded to the same carbon can form an exocyclic double bond. In another embodiment, two R⁸ groups bonded to the same carbon can form a carbonyl group.

In an alternative embodiment, the invention is directed to compounds or the use of such compounds of Formula VI:

-   wherein R, R¹, R², R³, R⁴, R⁵, R⁶, R^(x), Z, m, n, and y are as     defined above; -   each R¹⁴ is independently H, C₁-C₃ alkyl (including methyl) or     haloalkyl, cycloalkyl or cycloalkyl containing one or more     heteroatoms selected from N, O or S; -(alkylene)_(m)-C₃-C₈     cycloalkyl, -(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,     -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,     -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,     -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any     of which, other than heterocyclo, may be optionally independently     substituted with one or more R^(x) groups as allowed by valence, and     wherein two R^(x) groups bound to the same or adjacent atoms may     optionally combine to form a ring; -   or two R¹⁴ groups bonded to the same carbon can form an exocyclic     double bond; -   or two R¹⁴ groups bonded to the same carbon can form a carbonyl     group; and when the compound of Formula VI has a double bond, as     indicated by the (----), in the 6-membered ring fused to the     pyrimidine ring, two R¹⁴ groups are present as allowed for in     Formula VI above; or -   when the compound of Formula VI does not include a double bond, as     indicated by the (----), in the 6-membered ring fused to the     pyrimidine ring, four R¹⁴ groups are present as allowed for in     Formula VI above; -   wherein each heteroaryl is an aryl ring system that contains one or     more heteroatoms selected from the group O, N and S, wherein the     ring nitrogen and sulfur atom(s) are optionally oxidized, and     nitrogen atom(s) are optionally quarternized; -   wherein each aryl is a carbocyclic aromatic system containing one or     two rings, wherein such rings may be attached together in a fused     manner, and wherein each aryl may have 1 or more R^(x) substituents; -   wherein each heterocyclo is a saturated or partially saturated     heteroatom-containing ring radical, where the heteroatoms may be     selected from nitrogen, sulfur and oxygen, wherein each heterocyclo     is a monocyclic 6-8 membered ring or a 5-16 membered bicyclic ring     system, and wherein each heterocyclo may have 1 to 3 R^(x)     substituents;     or a pharmaceutically acceptable salt, prodrug or isotopic variant,     for example, partially or fully deuterated form thereof.

In some aspects, the compound is of Formula I or Formula II and R⁶ is absent.

In some aspects, the compound is of Formula III:

and the variables are as defined for compounds of Formulae I and II and pharmaceutically acceptable salts thereof.

In some aspects, R^(x) is not further substituted.

In some aspects, R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any of which may be optionally independently substituted with one or more R^(x) groups as allowed by valence, and wherein two R^(x) groups bound to the same or adjacent atom may optionally combine to form a ring and wherein m is 0 or 1 and n is 0, 1 or 2.

In some aspects, R⁸ is hydrogen or C₁-C₃ alkyl.

In some aspects, R is hydrogen or C₁-C₃ alkyl.

In some aspects, R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴, -(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ any of which may be optionally independently substituted with one or more R^(x) groups as allowed by valence, and wherein two R^(x) groups bound to the same or adjacent atom may optionally combine to form a ring.

In some aspects, R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴, -(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ without further substitution.

In some aspects, m in R² is 1. In a further aspect, the alkylene in R² is methylene.

In some aspects, R² is

wherein:

-   R^(2*) is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,     -(alkylene)_(m)-C(O)-(alkylene)_(m)-,     -(alkylene)_(m)-S(O)₂-(alkylene)_(m)- or     -(alkylene)_(m)-NH-(alkylene)_(m)- wherein each m is independently 0     or 1; -   P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl     group; -   each R^(x1) is independently     -(alkylene)_(m)-(C(O))_(m)-(alkylene)_(m)-(N(R^(N)))_(m)-(alkyl)_(m)     wherein each m is independently 0 or 1 provided at least one m is 1,     —(C(O))—O-alkyl, -(alkylene)_(m)-cycloalkyl wherein m is 0 or 1,     —N(R^(N))-cycloalkyl, —C(O)-cycloalkyl, -(alkylene)_(m)-heterocyclyl     wherein m is 0 or 1, or —N(R^(N))-heterocyclyl, —C(O)-heterocyclyl,     —S(O)₂-(alkylene)_(m) wherein m is 1 or 2, wherein:

R^(N) is H, C₁ to C₄ alkyl or C₁ to C₆ heteroalkyl, and

wherein two R^(x1) can, together with the atoms to which they attach on P, which may be

the same atom, form a ring; and

-   t is 0, 1 or 2.

In some aspects, each R^(x1) is only optionally substituted by unsubstituted alkyl, halogen or hydroxy.

In some aspects, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl.

In some aspects, at least one R^(x1) is -(alkylene)_(m)-heterocyclyl wherein m is 0 or 1.

In some aspects, R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some aspects, R² is

In some aspects, R² is

In some aspects, R² is

wherein:

-   R^(2*) is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,     -(alkylene)_(m)-C(O)-(alkylene)_(m)-,     -(alkylene)_(m)-S(O)₂-(alkylene)_(m)- or     -(alkylene)_(m)-NH-(alkylene)_(m)- wherein each m is independently 0     or 1; -   P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl     group; -   P1 is a 4- to 6-membered monocyclic saturated heterocyclyl group; -   each R^(x2) is independently hydrogen or alkyl; and -   s is 0, 1 or 2.

In some aspects, R² is

In some aspects, P1 includes at least one nitrogen.

In some aspects, any alkylene in R²* in any previous aspect is not further substituted.

In some aspects, R² is selected from the structures depicted in FIGS. 2-4.

In some aspects, R² is

In some aspects, the compound has general Formula I and more specifically one of the general structures disclosed in FIGS. 5A-9F wherein the variables are as previously defined.

In some aspects, the compound has general Formula Ia:

wherein R¹, R², R, R⁸, X and y are as previously defined.

In some embodiments, the compound has Formula Ia and R is alkyl.

In some embodiments, the compound has Formula Ia and R is H.

In some embodiments, the compound has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl and R^(2*) is as previously defined.

In some embodiments, the compound has Formula Ib:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ib and R is alkyl.

In some embodiments, the compound has Formula Ib and R is H.

In some embodiments, the compound has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is as previously defined.

In some embodiments, the compound has Formula Ic:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ic and R is alkyl.

In some embodiments, the compound has Formula Ic and R is H.

In some embodiments, the compound has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is as previously defined.

In some embodiments, the compound has Formula Id:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Id and R is alkyl.

In some embodiments, the compound has Formula Id and R is H.

In some embodiments, the compound has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is as previously defined.

In some embodiments, the compound has Formula Ie:

wherein R, R² and R⁸ are as previously defined. In some embodiments, the compound has Formula Ie and R is alkyl.

In some embodiments, the compound has Formula Ie and R is H.

In some embodiments, the compound has Formula Ie and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ie and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is as previously defined.

In some embodiments, the compound has Formula If:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula If and R is alkyl.

In some embodiments, the compound has Formula If and R is H.

In some embodiments, the compound has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is as previously defined.

In some embodiments, the compound has Formula Ig:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ig and R is alkyl.

In some embodiments, the compound has Formula Ig and R is H.

In some embodiments, the compound has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is as previously defined.

In some embodiments, the compound has Formula Ih:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ih and R is alkyl.

In some embodiments, the compound has Formula Ih and R is H.

In some embodiments, the compound has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is as previously defined.

In some embodiments, the compound has Formula Ii:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ii and R is alkyl.

In some embodiments, the compound has Formula Ii and R is H.

In some embodiments, the compound has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is as previously defined.

In some embodiments, the compound has Formula Ij:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ij and R is alkyl.

In some embodiments, the compound has Formula Ij and R is H.

In some embodiments, the compound has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some embodiments, the compound has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Ij and R is H, and X is CH and N.

In some embodiments, the compound has the structure:

In some embodiments, the compound has the structure Ik:

In some embodiments, the compound has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some embodiments, the compound has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Il:

In some embodiments, the compound has Formula Il and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some embodiments, the compound has Formula Il and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Im:

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Ha:

In some embodiments, the compound has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some embodiments, the compound has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula IIb:

In some embodiments, the compound has Formula IIb and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some embodiments, the compound has Formula IIb and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some aspects, the active compound is:

Further specific compounds that fall within the present invention and that can be used in the disclosed methods of treatment and compositions include, but are not limited to, the structures listed in Table 1 below.

TABLE 1 Structures of Tricyclic Lactams Structure Reference Structure A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

AA

BB

CC

DD

EE

FF

GG

HH

II

JJ

KK

LL

MM

NN

OO

PP

QQ

RR

SS

TT

UU

VV

WW

XX

YY

ZZ

AAA

BBB

CCC

DDD

EEE

FFF

GGG

HHH

III

JJJ

KKK

LLL

MMM

NNN

OOO

PPP

QQQ

RRR

SSS

TTT

UUU

VVV

WWW

XXX

YYY

ZZZ

AAAA

BBBB

CCCC

DDDD

EEEE

FFFF

GGGG

HHHH

Isotopic Substitution

The present invention includes compounds and the use of compounds with desired isotopic substitutions of atoms, at amounts above the natural abundance of the isotope, i.e., enriched. Isotopes are atoms having the same atomic number but different mass numbers, i.e., the same number of protons but a different number of neutrons. By way of general example and without limitation, isotopes of hydrogen, for example, deuterium (²H) and tritium (³H) may be used anywhere in described structures. Alternatively or in addition, isotopes of carbon, e.g., ¹³C and ¹⁴C, may be used. A preferred isotopic substitution is deuterium for hydrogen at one or more locations on the molecule to improve the performance of the drug. The deuterium can be bound in a location of bond breakage during metabolism (an α-deuterium kinetic isotope effect) or next to or near the site of bond breakage (a β-deuterium kinetic isotope effect).

Substitution with isotopes such as deuterium can afford certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements. Substitution of deuterium for hydrogen at a site of metabolic break down can reduce the rate of or eliminate the metabolism at that bond. At any position of the compound that a hydrogen atom may be present, the hydrogen atom can be any isotope of hydrogen, including protium (¹H), deuterium (²H) and tritium (³H). Thus, reference herein to a compound encompasses all potential isotopic forms unless the context clearly dictates otherwise.

The term “isotopically-labeled” analog refers to an analog that is a “deuterated analog”, a “¹³C-labeled analog,” or a “deuterated/¹³C-labeled analog.” The term “deuterated analog” means a compound described herein, whereby a H-isotope, i.e., hydrogen/protium (¹H), is substituted by a H-isotope, i.e., deuterium (²H). Deuterium substitution can be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted by at least one deuterium. In certain embodiments, the isotope is 90, 95 or 99% or more enriched in an isotope at any location of interest. In some embodiments it is deuterium that is 90, 95 or 99% enriched at a desired location.

Rb-positive Cancers and Proliferative Disorders

In particular, the active compounds described herein can be used to treat a subject suffering from an Rb-positive cancer or other Rb-positive abnormal cellular proliferative disorder. In some embodiments, the cancer or cellular proliferation disorder is a CDK4/6-replication dependent cancer or cellular proliferation disorder, which refers to a cancer or cellular proliferation disorder that requires the activity of CDK4/6 for replication or proliferation, or which may be growth inhibited through the activity of a selective CDK4/6 inhibitor. Cancers and disorders of such type can be characterized by (e.g., that has cells that exhibit) the presence of a functional Retinoblastoma protein. Such cancers and disorders are classified as being Rb-positive. Rb-positive abnormal cellular proliferation disorders, and variations of this term as used herein, refer to disorders or diseases caused by uncontrolled or abnormal cellular division which are characterized by the presence of a functional Retinoblastoma protein, which can include cancers. In one aspect of the present invention, the compounds and methods described herein can be used to treat a non-cancerous Rb-positive abnormal cellular proliferation disorder.

Targeted cancers suitable for administration of a compound described herein may include Rb-positive: estrogen-receptor positive, HER2-negative advanced breast cancer, late-line metastatic breast cancer, liposarcoma, non-small cell lung cancer, liver cancer, ovarian cancer, glioblastoma, refractory solid tumors, retinoblastoma positive breast cancer as well as retinoblastoma positive endometrial, vaginal and ovarian cancers and lung and bronchial cancers, adenocarcinoma of the colon, adenocarcinoma of the rectum, central nervous system germ cell tumors, teratomas, estrogen receptor-negative breast cancer, estrogen receptor-positive breast cancer, familial testicular germ cell tumors, HER2-negative breast cancer, HER2-positive breast cancer, male breast cancer, ovarian immature teratomas, ovarian mature teratoma, ovarian monodermal and highly specialized teratomas, progesterone receptor-negative breast cancer, progesterone receptor-positive breast cancer, recurrent breast cancer, recurrent colon cancer, recurrent extragonadal germ cell tumors, recurrent extragonadal non-seminomatous germ cell tumor, recurrent extragonadal seminomas, recurrent malignant testicular germ cell tumors, recurrent melanomas, recurrent ovarian germ cell tumors, recurrent rectal cancer, stage III extragonadal non-seminomatous germ cell tumors, stage III extragonadal seminomas, stage III malignant testicular germ cell tumors, stage III ovarian germ cell tumors, stage IV breast cancers, stage IV colon cancers, stage IV extragonadal non-seminomatous germ cell tumors, stage IV extragonadal seminoma, stage IV melanomas, stage IV ovarian germ cell tumors, stage IV rectal cancers, testicular immature teratomas, testicular mature teratomas. In particular embodiments, the targeted cancers included estrogen-receptor positive, HER2-negative advanced breast cancer, late-line metastatic breast cancer, liposarcoma, non-small cell lung cancer, liver cancer, ovarian cancer, glioblastoma, refractory solid tumors, retinoblastoma positive breast cancer as well as retinoblastoma positive endometrial, vaginal and ovarian cancers and lung and bronchial cancers, metastatic colorectal cancer, metastatic melanoma with CDK4 mutation or amplification, or cisplatin-refractory, unresectable germ cell tumors.

In one embodiment, the Rb-positive cancer is selected from an Rb-positive carcinoma, sarcoma, including, but not limited to, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, or a combination of one or more of the foregoing cancers.

In one embodiment, the Rb-positive cancer is selected from the group consisting of Rb-positive: fibrosarcoma, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, lymphangiosarcoma. Mesothelioma, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma; epidermoid carcinoma, malignant skin adnexal tumors, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, hypernephroma, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, glioma anaplastic; glioblastoma multiforme-neuroblastoma, medulloblastoma, malignant meningioma, malignant schwannoma, neurofibrosarcoma, parathyroid carcinoma, medullary carcinoma of thyroid, bronchial carcinoid, pheochromocytoma, Islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phylloide, salivary cancers, thymic carcinomas, bladder cancer, and Wilms tumor.

The presence or normal functioning of the retinoblastoma (Rb) tumor suppressor protein (Rb-positive) can be determined through any of the standard assays known to one of ordinary skill in the art, including but not limited to Western Blot, ELISA (enzyme linked immunoadsorbent assay), IHC (immunohistochemistry), and FACS (fluorescent activated cell sorting). The selection of the assay will depend upon the tissue, cell line or surrogate tissue sample that is utilized e.g., for example Western Blot and ELISA may be used with any or all types of tissues, cell lines or surrogate tissues, whereas the IHC method would be more appropriate wherein the tissue utilized in the methods of the present invention was a tumor biopsy. FACs analysis would be most applicable to samples that were single cell suspensions such as cell lines and isolated peripheral blood mononuclear cells. See for example, US 20070212736 “Functional Immunohistochemical Cell Cycle Analysis as a Prognostic Indicator for Cancer”. Alternatively, molecular genetic testing may be used for determination of retinoblastoma gene status. Molecular genetic testing for retinoblastoma includes the following as described in Lohmann and Gallie “Retinoblastoma. Gene Reviews” (2010) http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=retinoblastoma or Parsam et al. “A comprehensive, sensitive and economical approach for the detection of mutations in the RB 1 gene in retinoblastoma” Journal of Genetics, 88(4), 517-527 (2009).

In some embodiments, the cancer to be treated is selected from estrogen-receptor positive, HER2-negative advanced breast cancer, late-line metastatic breast cancer, liposarcoma, non-small cell lung cancer, liver cancer, ovarian cancer, glioblastoma, refractory solid tumors, retinoblastoma positive breast cancer as well as retinoblastoma positive endometrial, vaginal and ovarian cancers and lung and bronchial cancers.

CDK-Replication Dependent Cells and Cyclin-Dependent Kinase Inhibitors

Tissue-specific stem cells and subsets of other resident proliferating cells are capable of self-renewal, meaning that they are capable of replacing themselves throughout the adult mammalian lifespan through regulated replication. Additionally, stem cells divide asymmetrically to produce “progeny” or “progenitor” cells that in turn produce various components of a given organ. For example, in the hematopoietic system, the hematopoietic stem cells give rise to progenitor cells which in turn give rise to all the differentiated components of blood (e.g., white blood cells, red blood cells, and platelets) (see FIG. 1).

It has been found that certain proliferating cells, such as HSPCs, require the enzymatic activity of the proliferative kinases cyclin-dependent kinase 4 (CDK4) and/or cyclin-dependent kinase 6 (CDK6) for cellular replication. In contrast, the majority of proliferating cells in adult mammals (e.g., the more differentiated blood-forming cells in the bone marrow) do not require the activity of CDK4 and/or CDK6 (i.e., CDK4/6). These differentiated cells can proliferate in the absence of CDK4/6 activity by using other proliferative kinases, such as cyclin-dependent kinase 2 (CDK2) or cyclin-dependent kinase 1 (CDK1).

The present invention includes methods of treating certain cancers, in particular Rb-positive cancers, while minimizing the deleterious effects on CDK4/6-replication dependent healthy cells in a subject, and in particular, hematopoietic cells and/or progenitor cells (HSPCs), by the administration of a compound described herein to treat a specific Rb-positive cancer. In certain embodiments, the compound administered is selected from the group consisting of a compound or composition comprising Formula I, Formula II, Formula III, Formula IV, Formula V, or Formula VI, or a combination thereof. In certain embodiments, the compound administered is selected from the group consisting of a compound selected from Table 1.

In certain aspects, compounds, methods, and compositions are provided as chemotherapeutics which reduce or limit the deleterious effect of CDK4/6 inhibition on CDK4/6-replication dependent healthy cells in a subject undergoing CDK4/6 inhibitory treatment for an Rb-positive cancer. In certain embodiments, the compound administered is selected from the group consisting of the compound or a composition comprising Formula I, Formula II, Formula III, Formula IV, Formula V, or Formula VI, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In certain embodiments, the compound administered is selected from a compound contained in Table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the CDK4/6-replication dependent cells are hematopoietic stem cells and/or progenitor cells (HSPCs).

In certain embodiments, the compound administered is selected from the group consisting of the compound or a composition comprising Formula I, Formula II, Formula III, Formula IV, Formula V, or Formula VI, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof, or compound contained in Table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof, wherein the effect of the compound is short term and transient in nature, allowing a significant portion of the CDK4/6-replication dependent healthy cells to synchronously renter the cell-cycle quickly, for example within less than about 24, 30, 36, or 40 hours of the last administration of the compound.

In one embodiment, a compound useful in the methods described herein is a selective CDK4/6 inhibitor compound that selectively inhibits at least one of CDK4 and CDK6 or through the inhibition of cellular replication of an Rb-positive cancer. In one embodiment, the tricyclic lactam compounds for use in the described methods are CDK4/6 inhibitors, with minimal CDK2 inhibitory activity. In one embodiment, a compound for use in the methods described herein has a CDK4/CycD1 IC₅₀ inhibitory concentration value that is >100, >200, >300, >400, >500, >600, >700, >800, >900, >1000, >1250, >1500 times, >1800 times, >2000 times, >2200 times, >2500 times, >2700 times, >3000 times, >3200 times lower than its respective IC₅₀ concentration value for CDK2/CycE inhibition. In one embodiment, a compound for use in the methods described herein has an IC₅₀ concentration value for CDK4/CycD1 inhibition that is about <1.50 nM, <1.25 nM, <1.0 nM, <0.90 nM, <0.85 nM, <0.80 nM, <0.75 nM, <0.70 nM, <0.65 nM, <0.60 nM, <0.55 nM, or less. In one embodiment, a tricyclic lactam for use in the methods described herein has an IC₅₀ concentration value for CDK2/CycE inhibition that is about >1.0 μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0 μM, >2.25 μM, >2.50 μM, >2.75 μM, >3.0 μM, >3.25 μM, >3.5 μM or greater. In one embodiment, a compound for use in the methods described herein has an IC₅₀ concentration value for CDK2/CycA IC₅₀ that is >0.80 μM, >0.85 μM, >0.90 μM, >0.95 μM, >0.1.0 μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0 μM, >2.25 μM, >2.50 μM, >2.75 uM, >3.0 μM or greater.

In one embodiment, the compounds described herein are used in CDK4/6-replication dependent healthy cell cycling strategies wherein a subject is exposed to regular, repeated chemotherapeutic treatments for an Rb-positive cancer. Such cycling allows CDK4/6-replication dependent cells to regenerate damaged blood cell lineages between regular, repeated treatments, and reduces the risk associated with long term CDK4/6 inhibition.

In one embodiment, the use of a compound described herein provides for a rapid, reentry into the cell cycle by CDK4/6-replication dependent healthy cells, for example HSPCs, so that a portion of the cells exhibit a level of cell cycle activity or are capable of entering the cell cycle and proliferate during a continuous treatment regime, for example, a treatment regime wherein the compound is administered for an extended period, for example, 5 continuous days, 7 continuous days, 10 continuous days, 14 continuous days, 18 continuous days, 21 continuous days, 24 continuous days, 28 continuous days, 35 continuous days or more. In one embodiment, a compound useful in a described method is administered for a continuous period, for example, 21, 28, 35 days or more, without the requirement for an off-cycle period or drug holiday. In one embodiment, the use of a compound described herein eliminates the need for an off-cycle period, drug holiday, or reduction in co-administered anti-neoplastic compound concentration during treatment.

According to the present invention, a compound described herein can be administered as a chemotherapeutic to a subject having an Rb-positive proliferation disorder on any treatment schedule and in any dose consistent with the prescribed course of treatment. For instance the compound can be administered once a day, twice a day or three times a day. The compound can be administered on alternating days, or every third day, or every fourth day, or every fifth day, or every sixth day or once a week. The compound can be administered every other week or monthly.

Combination Therapy

In one aspect of the invention, the compounds disclosed herein can be beneficially administered in combination with another therapeutic regimen for beneficial, additive or synergystic effect.

In one embodiment, a compound/method of the present invention is used in combination with another therapy to treat the Rb-positive cancer. The second therapy can be an immunotherapy. As discussed in more detail below, the compound can be conjugated to an antibody, radioactive agent, or other targeting agent that directs the compound to the diseased or abnormally proliferating cell. In another embodiment, the compound is used in combination with another pharmaceutical or a biologic agent (for example an antibody) to increase the efficacy of treatment with a combined or a synergistic approach. In an embodiment, the compound can be used with T-cell vaccination, which typically involves immunization with inactivated autoreactive T cells to eliminate an Rb-positive cancer cell population as described herein. In another embodiment, the compound is used in combination with a bispecific T-cell Engager (BiTE), which is an antibody designed to simultaneously bind to specific antigens on endogenous T cells and Rb-positive cancer cells as described herein, linking the two types of cells.

In one embodiment, the additional therapy is a monoclonal antibody (MAb). Some MAbs stimulate an immune response that destroys cancer cells. Similar to the antibodies produced naturally by B cells, these MAbs “coat” the cancer cell surface, triggering its destruction by the immune system. For example, bevacizumab targets vascular endothelial growth factor(VEGF), a protein secreted by tumor cells and other cells in the tumor's microenvironment that promotes the development of tumor blood vessels. When bound to bevacizumab, VEGF cannot interact with its cellular receptor, preventing the signaling that leads to the growth of new blood vessels. Similarly, cetuximab and panitumumab target the epidermal growth factor receptor (EGFR), and trastuzumab targets the human epidermal growth factor receptor 2 (HER-2). MAbs that bind to cell surface growth factor receptors prevent the targeted receptors from sending their normal growth-promoting signals. They may also trigger apoptosis and activate the immune system to destroy tumor cells.

Another group of cancer therapeutic MAbs are the immunoconjugates. These MAbs, which are sometimes called immunotoxins or antibody-drug conjugates, consist of an antibody attached to a cell-killing substance, such as a plant or bacterial toxin, a chemotherapy drug, or a radioactive molecule. The antibody latches onto its specific antigen on the surface of a cancer cell, and the cell-killing substance is taken up by the cell. FDA-approved conjugated MAbs that work this way include ado-trastuzumab emtansine, which targets the HER-2 molecule to deliver the drug DM1, which inhibits cell proliferation, to HER-2 expressing metastatic breast cancer cells.

Immunotherapies with T cells engineered to recognize cancer cells via bispecific antibodies (bsAbs) or chimeric antigen receptors (CARs) are approaches with potential to ablate both dividing and non/slow-dividing subpopulations of cancer cells.

Bispecific antibodies, by simultaneously recognizing target antigen and an activating receptor on the surface of an immune effector cell, offer an opportunity to redirect immune effector cells to kill cancer cells. The other approach is the generation of chimeric antigen receptors by fusing extracellular antibodies to intracellular signaling domains. Chimeric antigen receptor-engineered T cells are able to specifically kill tumor cells in a MHC-independent way.

In some embodiments, the compound can be administered to the subject in combination with other chemotherapeutic agents. If convenient, the compounds described herein can be administered at the same time as another chemotherapeutic agent, in order to simplify the treatment regimen. In some embodiments, the compound and the other chemotherapeutic can be provided in a single formulation. In one embodiment, the use of the compounds described herein is combined in a therapeutic regime with other agents. Such agents may include, but are not limited to, tamoxifen, midazolam, letrozole, bortezomib, anastrozole, goserelin, an mTOR inhibitor, a PI3 kinase inhibitors, dual mTOR-PI3K inhibitors, MEK inhibitors, RAS inhibitors, ALK inhibitors, HSP inhibitors (for example, HSP70 and HSP 90 inhibitors, or a combination thereof), BCL-2 inhibitors, apoptotic inducing compounds, AKT inhibitors, including but not limited to, MK-2206, GSK690693, Perifosine, (KRX-0401), GDC-0068, Triciribine, AZD5363, Honokiol, PF-04691502, and Miltefosine, PD-1 inhibitors including but not limited to, Nivolumab, CT-011 (pidilizumab), MK-3475 (pembrolizumab), BMS936558, MPDL328OA (Roche), and AMP-514 or FLT-3 inhibitors, including but not limited to, P406, Dovitinib, Quizartinib (AC220), Amuvatinib (MP-470), Tandutinib (MLN518), ENMD-2076, and KW-2449, or combinations thereof. Examples of mTOR inhibitors include but are not limited to rapamycin and its analogs, everolimus (Afinitor), temsirolimus, ridaforolimus, sirolimus, and deforolimus. Examples of P13 kinase inhibitors include but are not limited to Wortmannin, demethoxyviridin, perifosine, idelalisib, PX-866, IPI-145, BAY 80-6946, BEZ235, RP6503, TGR 1202 (RP5264), MLN1117 (INK1117), Pictilisib, Buparlisib, SAR245408 (XL147), SAR245409 (XL765), Palomid 529, ZSTK474, PWT33597, RP6530, CUDC-907, and AEZS-136. Examples of MEK inhibitors include but are not limited to Tametinib, Selumetinib, MEK162, GDC-0973 (XL518), and PD0325901. Examples of RAS inhibitors include but are not limited to Reolysin and siGl2D LODER. Examples of ALK inhibitors include but are not limited to Crizotinib, AP26113, and LDK378. HSP inhibitors include but are not limited to Geldanamycin or 17-N-Allylamino-17-demethoxygeldanamycin (17AAG), and Radicicol. In a particular embodiment, a compound described herein is administered in combination with letrozole and/or tamoxifen. Other chemotherapeutic agents that can be used in combination with the compounds described herein include, but are not limited to, chemotherapeutic agents that do not require cell cycle activity for their anti-neoplastic effect.

In one embodiment, a CDK4/6 inhibitor described herein can be combined with a chemotherapeutic selected from, but are not limited to, Imatinib mesylate (Gleevac®), Dasatinib (Sprycel®), Nilotinib (Tasigna®), Bosutinib (Bosulif®), Trastuzumab (Herceptin®), Pertuzumab (Perjeta™), Lapatinib (Tykerb®), Gefitinib (Iressa®), Erlotinib (Tarceva®), Cetuximab (Erbitux®), Panitumumab (Vectibix®), Vandetanib (Caprelsa®), Vemurafenib (Zelboraf®), Vorinostat (Zolinza®), Romidepsin (Istodax®), Bexarotene (Tagretin®), Alitretinoin (Panretin®), Tretinoin (Vesanoid®), Carfilizomib (Kyprolis™), Pralatrexate (Folotyn®), Bevacizumab (Avastin®), Ziv-aflibercept (Zaltrap®), Sorafenib (Nexavar®), Sunitinib (Sutent®), Pazopanib (Votrient®), Regorafenib (Stivarga®), and Cabozantinib (Cometriq™).

In certain aspects, the additional therapeutic agent is an anti-inflammatory agent, a chemotherapeutic agent, a radiotherapeutic, additional therapeutic agents, or immunosuppressive agents.

Suitable chemotherapeutic agents include, but are not limited to, radioactive molecules, toxins, also referred to as cytotoxins or cytotoxic agents, which includes any agent that is detrimental to the viability of cells, agents, and liposomes or other vesicles containing chemotherapeutic compounds. General anticancer pharmaceutical agents include: Vincristine (Oncovin®) or liposomal vincristine (Marqibo®), Daunorubicin (daunomycin or Cerubidine®) or doxorubicin (Adriamycin®), Cytarabine (cytosine arabinoside, ara-C, or Cytosar®), L-asparaginase (Elspar®) or PEG-L-asparaginase (pegaspargase or Oncaspar®), Etoposide (VP-16), Teniposide (Vumon®), 6-mercaptopurine (6-MP or Purinethol®), Methotrexate, Cyclophosphamide (Cytoxan®), Prednisone, Dexamethasone (Decadron), imatinib (Gleevec®), dasatinib (Sprycel®), nilotinib (Tasigna®), bosutinib (Bosulif®), and ponatinib (Iclusig™) Examples of additional suitable chemotherapeutic agents include but are not limited to 1-dehydrotestosterone, 5-fluorouracil decarbazine, 6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin, aldesleukin, alkylating agents, allopurinol sodium, altretamine, amifostine, anastrozole, anthramycin (AMC)), anti-mitotic agents, cis-dichlorodiamine platinum (II) (DDP) cisplatin), diamino dichloro platinum, anthracyclines, antibiotics, antimetabolites, asparaginase, BCG live (intravesical), betamethasone sodium phosphate and betamethasone acetate, bicalutamide, bleomycin sulfate, busulfan, calcium leucouorin, calicheamicin, capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU), Chlorambucil, Cisplatin, Cladribine, Colchicin, conjugated estrogens, Cyclophosphamide, Cyclothosphamide, Cytarabine, Cytarabine, cytochalasin B, Cytoxan, Dacarbazine, Dactinomycin, dactinomycin (formerly actinomycin), daunirubicin HCL, daunorucbicin citrate, denileukin diftitox, Dexrazoxane, Dibromomannitol, dihydroxy anthracin dione, Docetaxel, dolasetron mesylate, doxorubicin HCL, dronabinol, E. coli L-asparaginase, emetine, epoetin-α, Erwinia L-asparaginase, esterified estrogens, estradiol, estramustine phosphate sodium, ethidium bromide, ethinyl estradiol, etidronate, etoposide citrororum factor, etoposide phosphate, filgrastim, floxuridine, fluconazole, fludarabine phosphate, fluorouracil, flutamide, folinic acid, gemcitabine HCL, glucocorticoids, goserelin acetate, gramicidin D, granisetron HCL, hydroxyurea, idarubicin HCL, ifosfamide, interferon α-2b, irinotecan HCL, letrozole, leucovorin calcium, leuprolide acetate, levamisole HCL, lidocaine, lomustine, maytansinoid, mechlorethamine HCL, medroxyprogesterone acetate, megestrol acetate, melphalan HCL, mercaptipurine, mesna, methotrexate, methyltestosterone, mithramycin, mitomycin C, mitotane, mitoxantrone, nilutamide, octreotide acetate, ondansetron HCL, paclitaxel, pamidronate disodium, pentostatin, pilocarpine HCL, plimycin, polifeprosan 20 with carmustine implant, porfimer sodium, procaine, procarbazine HCL, propranolol, rituximab, sargramostim, streptozotocin, tamoxifen, taxol, teniposide, tenoposide, testolactone, tetracaine, thioepa chlorambucil, thioguanine, thiotepa, topotecan HCL, toremifene citrate, trastuzumab, tretinoin, valrubicin, vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate.

Additional therapeutic agents that can be administered in combination with a compound disclosed herein can include bevacizumab, sutinib, sorafenib, 2-methoxyestradiol or 2ME2, finasunate, vatalanib, vandetanib, aflibercept, volociximab, etaracizumab (MEDI-522), cilengitide, erlotinib, cetuximab, panitumumab, gefitinib, trastuzumab, dovitinib, figitumumab, atacicept, rituximab, alemtuzumab, aldesleukine, atlizumab, tocilizumab, temsirolimus, everolimus, lucatumumab, dacetuzumab, HLL1, huN901-DM1, atiprimod, natalizumab, bortezomib, carfilzomib, marizomib, tanespimycin, saquinavir mesylate, ritonavir, nelfinavir mesylate, indinavir sulfate, belinostat, panobinostat, mapatumumab, lexatumumab, dulanermin, ABT-737, oblimersen, plitidepsin, talmapimod, P276-00, enzastaurin, tipifarnib, perifosine, imatinib, dasatinib, lenalidomide, thalidomide, simvastatin, and celecoxib.

In one aspect of the present invention, a compound described herein can be combined with at least one immunosuppressive agent. The immunosuppressive agent is preferably selected from the group consisting of a calcineurin inhibitor, e.g. a cyclosporin or an ascomycin, e.g. Cyclosporin A (NEORAL®), FK506 (tacrolimus), pimecrolimus, a mTOR inhibitor, e.g. rapamycin or a derivative thereof, e.g. Sirolimus (RAPAMUNE®), Everolimus (Certican®), temsirolimus, zotarolimus, biolimus-7, biolimus-9, a rapalog, e.g.ridaforolimus, azathioprine, campath 1H, a S1P receptor modulator, e.g. fingolimod or an analogue thereof, an anti IL-8 antibody, mycophenolic acid or a salt thereof, e.g. sodium salt, or a prodrug thereof, e.g. Mycophenolate Mofetil (CELLCEPT®), OKT3 (ORTHOCLONE OKT3®), Prednisone, ATGAM®, THYMOGLOBULIN®, Brequinar Sodium, OKT4, T10B9.A-3A, 33B3.1, 15-deoxyspergualin, tresperimus, Leflunomide ARAVA®, CTLAI-Ig, anti-CD25, anti-IL2R, Basiliximab (SIMULECT®), Daclizumab (ZENAPAX®), mizorbine, methotrexate, dexamethasone, ISAtx-247, SDZ ASM 981 (pimecrolimus, Elidel®), CTLA4lg (Abatacept), belatacept, LFA3lg, etanercept (sold as Enbrel® by Immunex), adalimumab (Humira®), infliximab (Remicade®), an anti-LFA-1 antibody, natalizumab (Antegren®), Enlimomab, gavilimomab, antithymocyte immunoglobulin, siplizumab, Alefacept efalizumab, pentasa, mesalazine, asacol, codeine phosphate, benorylate, fenbufen, naprosyn, diclofenac, etodolac and indomethacin, aspirin and ibuprofen.

In one aspect of the present invention, a compound described herein can be combined with at least one anti-inflammatory agent. The anti-inflammatory agent can be a steroidal anti-inflammatory agent, a nonsteroidal anti-inflammatory agent, or a combination thereof. In some embodiments, anti-inflammatory drugs include, but are not limited to, alclofenac, alclometasone dipropionate, algestone acetonide, alpha amylase, amcinafal, amcinafide, amfenac sodium, amiprilose hydrochloride, anakinra, anirolac, anitrazafen, apazone, balsalazide disodium, bendazac, benoxaprofen, benzydamine hydrochloride, bromelains, broperamole, budesonide, carprofen, cicloprofen, cintazone, cliprofen, clobetasol propionate, clobetasone butyrate, clopirac, cloticasone propionate, cormethasone acetate, cortodoxone, deflazacort, desonide, desoximetasone, dexamethasone dipropionate, diclofenac potassium, diclofenac sodium, diflorasone diacetate, diflumidone sodium, diflunisal, difluprednate, diftalone, dimethyl sulfoxide, drocinonide, endrysone, enlimomab, enolicam sodium, epirizole, etodolac, etofenamate, felbinac, fenamole, fenbufen, fenclofenac, fenclorac, fendosal, fenpipalone, fentiazac, flazalone, fluazacort, flufenamic acid, flumizole, flunisolide acetate, flunixin, flunixin meglumine, fluocortin butyl, fluorometholone acetate, fluquazone, flurbiprofen, fluretofen, fluticasone propionate, furaprofen, furobufen, halcinonide, halobetasol propionate, halopredone acetate, ibufenac, ibuprofen, ibuprofen aluminum, ibuprofen piconol, ilonidap, indomethacin, indomethacin sodium, indoprofen, indoxole, intrazole, isoflupredone acetate, isoxepac, isoxicam, ketoprofen, lofemizole hydrochloride, lomoxicam, loteprednol etabonate, meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate, mefenamic acid, mesalamine, meseclazone, methylprednisolone suleptanate, morniflumate, nabumetone, naproxen, naproxen sodium, naproxol, nimazone, olsalazine sodium, orgotein, orpanoxin, oxaprozin, oxyphenbutazone, paranyline hydrochloride, pentosan polysulfate sodium, phenbutazone sodium glycerate, pirfenidone, piroxicam, piroxicam cinnamate, piroxicam olamine, pirprofen, prednazate, prifelone, prodolic acid, proquazone, proxazole, proxazole citrate, rimexolone, romazarit, salcolex, salnacedin, salsalate, sanguinarium chloride, seclazone, sermetacin, sudoxicam, sulindac, suprofen, talmetacin, talniflumate, talosalate, tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam, tesimide, tetrydamine, tiopinac, tixocortol pivalate, tolmetin, tolmetin sodium, triclonide, triflumidate, zidometacin, zomepirac sodium, aspirin (acetylsalicylic acid), salicylic acid, corticosteroids, glucocorticoids, tacrolimus, pimecorlimus, prodrugs thereof, co-drugs thereof, and combinations thereof

In one aspect of the present invention, a compound described herein can be combined with at least one immunomodulatory agent. In one embodiment, the immunomodulatory agent is selected from the group consisting of a CTLA-4 inhibitor, PD-1 or anti-PD-1 agent, IFN-alpha, IFN-beta, and a vaccine, for example, a cancer vaccine. In one embodiment, the PD-1 agent is Keytruda® (pembrolizumab). In one embodiment, the PD-1 agent is Opdivo (nivolumab). In one embodiment, the CTLA-4 inhibitor is Yervoy® (ipilimumab).

In certain embodiments, a compound described herein is administered to the subject prior to treatment with another chemotherapeutic agent, during treatment with another chemotherapeutic agent, after administration of another chemotherapeutic agent, or a combination thereof

In some embodiments, the compound can be administered to the subject such that the other chemotherapeutic agent can be administered either at higher doses (increased chemotherapeutic dose intensity) or more frequently (increased chemotherapeutic dose density). Dose-dense chemotherapy is a chemotherapy treatment plan in which drugs are given with less time between treatments than in a standard chemotherapy treatment plan. Chemotherapy dose intensity represents unit dose of chemotherapy administered per unit time. Dose intensity can be increased or decreased through altering dose administered, time interval of administration, or both.

In one embodiment of the invention, the compounds described herein can be administered in a concerted regimen with another agent such as a non-DNA-damaging, targeted anti-neoplastic agent or a hematopoietic growth factor agent. It has been recently reported that the untimely administration of hematopoietic growth factors can have serious side effects. For example, the use of the EPO family of growth factors has been associated with arterial hypertension, cerebral convulsions, hypertensive encephalopathy, thromboembolism, iron deficiency, influenza like syndromes and venous thrombosis. The G-CSF family of growth factors has been associated with spleen enlargement and rupture, respiratory distress syndrome, allergic reactions and sickle cell complications. By combining the administration of the short-lived selective compounds described herein and methods of the present invention with the timely administration of hematopoietic growth factors, for example, at the time point wherein the affected cells are no longer under growth arrest, it is possible for the health care practitioner to decrease the amount of the growth factor to minimize the unwanted adverse effects while achieving the desired therapeutic benefit. In one embodiment, the growth factor is administered upon cessation of the effect of the compound on the CDK4/6 replication dependent healthy cells, for example HSPCs. Thus, in this embodiment, the use of a selective compound described herein in an anti-neoplastic therapeutic regime may allow the subject to receive a reduced amount of growth factor because the targeted hematopoietic cells will have reentered the cell cycle quicker than treatment with other CDK4/6 inhibitors, for example PD0332991. In addition, rapid cell-cycle reentry following G1 arrest using a compound described herein provides for the ability to time the administration of hematopoietic growth factors to assist in the reconstitution of hematopoietic cell lines to maximize the growth factor effect, that is, when the growth factors will be most effective. As such, in one embodiment, the use of the compounds or methods described herein is combined with the use of hematopoietic growth factors including, but not limited to, granulocyte colony stimulating factor (G-CSF, for example, sold as Neupogen (filgrastin), Neulasta (peg-filgrastin), or lenograstin), granulocyte-macrophage colony stimulating factor (GM-CSF, for example sold as molgramostim and sargramostim (Leukine)), M-CSF (macrophage colony stimulating factor), thrombopoietin (megakaryocyte growth development factor (MGDF), for example sold as Romiplostim and Eltrombopag) interleukin (IL)-12, interleukin-3, interleukin-11 (adipogenesis inhibiting factor or oprelvekin), SCF (stem cell factor, steel factor, kit-ligand, or KL) and erythropoietin (EPO), and their derivatives (sold as for example epoetin-α as Darbopoetin, Epocept, Nanokine, Epofit, Epogin, Eprex and Procrit; epoetin-β sold as for example NeoRecormon, Recormon and Micera), epoetin-delta (sold as for example Dynepo), epoetin-omega (sold as for example Epomax), epoetin zeta (sold as for example Silapo and Reacrit) as well as for example Epocept, EPOTrust, Erypro Safe, Repoeitin, Vintor, Epofit, Erykine, Wepox, Espogen, Relipoeitin, Shanpoietin, Zyrop and EPIAO). In one embodiment, the tricyclic lactam is administered prior to administration of the hematopoietic growth factor. In one embodiment, the hematopoietic growth factor administration is timed so that the compound's effect on HSPCs has dissipated. In one embodiment, the growth factor is administered at least 20 hours after the administration of a compound described herein.

If desired, multiple doses of a compound described herein can be administered to the subject. Alternatively, the subject can be given a single dose of a compound described herein. In some embodiments, the CDK4/6-replication dependent healthy cells can be arrested for longer periods, for example, over a period of hours, days, weeks and/or months, through multiple, limited-time separated administrations of a compound described herein.

The reduction in side effects by the compounds described herein can allow for dose intensification (e.g., more therapy can be given in a fixed period of time), which will translate to better efficacy. Therefore, the presently disclosed methods can result in regimens that are less toxic and more effective. When appropriate, the small molecules can be formulated for oral, topical, intranasal, inhalation, intravenous or any other desired form of administration.

The use of a compound as described herein can induce selective G1 arrest in CDK4/6-dependent cells (e.g., as measured in a cell-based in vitro assay). In one embodiment, the tricyclic lactam is capable of increasing the percentage of CDK4/6-dependent cells in the G1 phase, while decreasing the percentage of CDK4/6-dependent cells in the G2/M phase and S phase. In one embodiment, the compound induces substantially pure (i.e., “clean”) G1 cell cycle arrest in the CDK4/6-dependent cells, e.g., wherein treatment with the compound induces cell cycle arrest such that the majority of cells are arrested in G1 as defined by standard methods (e.g. propidium iodide (PI) staining or others) with the population of cells in the G2/M and S phases combined being less than about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 3% or less of the total cell population. Methods of assessing the cell phase of a population of cells are known in the art (see, for example, in U.S. Patent Application Publication No. 2002/0224522) and include cytometric analysis, microscopic analysis, gradient centrifugation, elutriation, fluorescence techniques including immunofluorescence, and combinations thereof. Cytometric techniques include exposing the cell to a labeling agent or stain, such as DNA-binding dyes, e.g., PI, and analyzing cellular DNA content by flow cytometry. Immunofluorescence techniques include detection of specific cell cycle indicators such as, for example, thymidine analogs (e.g., 5-bromo-2-deoxyuridine (BrdU) or an iododeoxyuridine), with fluorescent antibodies.

In some embodiments, the use of a tricyclic lactam compound described herein results in reduced or substantially free of off-target effects, for example, related to inhibition of kinases other than CDK4 and or CDK6 such as CDK2. Furthermore, in certain embodiments, the use of the compounds described herein should not induce cell cycle arrest in CDK4/6-independent cells.

In one aspect of the invention, a compound disclosed herein can be beneficially administered in combination with any therapeutic regimen entailing radiotherapy, chemotherapy, or other therapeutic agents. In one embodiment, a tricyclic lactam compound described herein is administered to a subject with an Rb-positive cancer prior to ionizing radiation (IR) treatment, wherein the CDK4/6 inhibitory effect allows the cancer cells to be arrested in G0/G1 replication, and as the CDK4/6 inhibitory effect from the tricyclic lactam wears off prior to IR exposure, released to proliferate in a synchronized way, thus increasing the effectiveness of the IR treatment. In additional embodiments the compounds disclosed herein can be beneficially administered in combination with therapeutic agents targeting auto-immune disorders.

Drug Conjugates

In one embodiment, the activity of an active compound for a purpose described herein can be augmented through conjugation to an agent that targets the diseased or abnormally proliferating cell or otherwise enhances activity, delivery, pharmacokinetics or other beneficial property.

For example, the compound can be administered as an antibody-drug conjugates (ADC). In certain embodiments, a selected compound described herein can be administered in conjugation or combination with an antibody or antibody fragment. Fragments of an antibody can be produced through chemical or genetic mechanisms. The antibody fragment can be an antigen binding fragment. For example, the antigen binding fragment can be selected from an Fab, Fab′, (Fab′)2, or Fv. The antibody fragment can be a Fab. Monovalent F(ab) fragments have one antigen binding site. The antibody can be a divalent (Fab′)2 fragment, which has two antigen binding regions that are linked by disulfide bonds. In one embodiment, the antigen fragment is a (Fab′). Reduction of F(ab′)2 fragments produces two monovalent Fab′ fragments, which have a free sulfhydryl group that is useful for conjugation to other molecules.

A selected compound described herein can be administered in conjugation or combination with a Fv fragment. Fv fragments are the smallest fragment made from enzymatic cleavage of IgG and IgM class antibodies. Fv fragments have the antigen-binding site made of the VH and VC regions, but they lack the CH1 and CL regions. The VH and VL chains are held together in Fv fragments by non-covalent interactions.

In one embodiment, a selected compound as described herein can be administered in combination with an antibody fragment selected from the group consisting of an ScFv, domain antibody, diabody, triabody, tetrabody, Bis-scFv, minibody, Fab2, or Fab3 antibody fragment. In one embodiment, the antibody fragment is a ScFv. Genetic engineering methods allow the production of single chain variable fragments (ScFv), which are Fv type fragments that include the VH and VL domains linked with a flexible peptide When the linker is at least 12 residues long, the ScFv fragments are primarily monomeric. Manipulation of the orientation of the V-domains and the linker length creates different forms of Fv molecules. Linkers that are 3-11 residues long yield scFv molecules that are unable to fold into a functional Fv domain. These molecules can associate with a second scFv molecule, to create a bivalent diabody. In one embodiment, the antibody fragment administered in combination with a selected compound described herein is a bivalent diabody. If the linker length is less than three residues, scFv molecules associate into triabodies or tetrabodies. In one embodiment, the antibody fragment is a triabody. In one embodiment, the antibody fragment is a tetrabody. Multivalent scFvs possess greater functional binding affinity to their target antigens than their monovalent counterparts by having binding to two more target antigens, which reduces the off-rate of the antibody fragment. In one embodiment, the antibody fragment is a minibody. Minibodies are scFv-CH3 fusion proteins that assemble into bivalent dimers. In one embodiment, the antibody fragment is a Bis-scFv fragment. Bis-scFv fragments are bispecific. Miniaturized ScFv fragments can be generated that have two different variable domains, allowing these Bis-scFv molecules to concurrently bind to two different epitopes.

In one embodiment, a selected compound described herein is administered in conjugation or combination with a bispecific dimer (Fab2) or trispecific dimer (Fab3). Genetic methods are also used to create bispecific Fab dimers (Fab2) and trispecific Fab trimers (Fab3). These antibody fragments are able to bind 2 (Fab2) or 3 (Fab3) different antigens at once.

In one embodiment, a selected compound described herein is administered in conjugation or combination with an rIgG antibody fragment. rIgG antibody fragments refers to reduced IgG (75,000 daltons) or half-IgG. It is the product of selectively reducing just the hinge-region disulfide bonds. Although several disulfide bonds occur in IgG, those in the hinge-region are most accessible and easiest to reduce, especially with mild reducing agents like 2-mercaptoethylamine (2-MEA). Half-IgG are frequently prepared for the purpose of targeting the exposing hinge-region sulfhydryl groups that can be targeted for conjugation, either antibody immobilization or enzyme labeling.

In other embodiments, a selected active compound described herein can be linked to a radioisotope to increase efficacy, using methods well known in the art. Any radioisotope that is useful against Rb-positive cancer cells can be incorporated into the conjugate, for example, but not limited to, ¹³¹I, ¹²³I, ¹⁹²Ir, ³²P, ⁹⁰Sr, ¹⁹⁸Au, ²²⁶Ra, ⁹⁰Y, ²⁴¹Am, ²⁵²Cf, ⁶⁰Co, or ¹³⁷Cs.

Of note, the linker chemistry can be important to efficacy and tolerability of the drug conjugates. The thio-ether linked T-DM1 increases the serum stability relative to a disulfide linker version and appears to undergo endosomal degradation, resulting in intra-cellular release of the cytotoxic agent, thereby improving efficacy and tolerability, See, Barginear, M. F. and Budman, D. R., Trastuzumab-DM1: A review of the novel immune-conjugate for HER2-overexpressing breast cancer, The Open Breast Cancer Journal, 1:25-30, 2009.

Examples of early and recent antibody-drug conjugates, discussing drugs, linker chemistries and classes of targets for product development that may be used in the present invention can be found in the reviews by Casi, G. and Neri, D., Antibody-drug conjugates: basic concepts, examples and future perspectives, J. Control Release 161(2):422-428, 2012, Chari, R. V., Targeted cancer therapy: conferring specificity to cytotoxic drugs, Acc. Chem. Rev., 41(1):98-107, 2008, Sapra, P. and Shor, B., Monoclonal antibody-based therapies in cancer: advances and challenges, Pharmacol. Ther., 138(3):452-69, 2013, Schliemann, C. and Neri, D., Antibody-based targeting of the tumor vasculature, Biochim. Biophys. Acta., 1776(2):175-92, 2007, Sun, Y., Yu, F., and Sun, B. W., Antibody-drug conjugates as targeted cancer therapeutics, Yao Xue Xue Bao, 44(9):943-52, 2009, Teicher, B. A., and Chari, R. V., Antibody conjugate therapeutics: challenges and potential, Clin. Cancer Res., 17(20):6389-97, 2011, Firer, M. A., and Gellerman, G. J., Targeted drug delivery for cancer therapy: the other side of antibodies, J. Hematol. Oncol., 5:70, 2012, Vlachakis, D. and Kossida, S., Antibody Drug Conjugate bioinformatics: drug delivery through the letterbox, Comput. Math. Methods Med., 2013; 2013:282398, Epub 2013 Jun. 19, Lambert, J. M., Drug-conjugated antibodies for the treatment of cancer, Br. J. Clin. Pharmacol., 76(2):248-62, 2013, Concalves, A., Tredan, O., Villanueva, C. and Dumontet, C., Antibody-drug conjugates in oncology: from the concept to trastuzumab emtansine (T-DM1), Bull. Cancer, 99(12):1183-1191, 2012, Newland, A. M., Brentuximab vedotin: a CD-30-directed antibody-cytotoxic drug conjugate, Pharmacotherapy, 33(1):93-104, 2013, Lopus, M., Antibody-DM1 conjugates as cancer therapeutics, Cancer Lett., 307(2):113-118, 2011, Chu, Y. W. and Poison, A., Antibody-drug conjugates for the treatment of B-cell non-Hodgkin's lymphoma and leukemia, Future Oncol., 9(3):355-368, 2013, Bertholjotti, I., Antibody-drug conjugate—a new age for personalized cancer treatment, Chimia, 65(9): 746-748, 2011, Vincent, K. J., and Zurini, M., Current strategies in antibody engineering: Fc engineering and pH—dependent antigen binding, bispecific antibodies and antibody drug conjugates, Biotechnol. J., 7(12):1444-1450, 2012, Haeuw, J. F., Caussanel, V., and Beck, A., Immunoconjugates, drug-armed antibodies to fight against cancer, Med. Sci., 25(12):1046-1052, 2009 and Govindan, S. V., and Goldenberg, D. M., Designing immunoconjugates for cancer therapy, Expert Opin. Biol. Ther., 12(7):873-890, 2012.

Pharmaceutical Compositions and Dosage Forms

An active compound described herein, or its salt, isotopic analog, or prodrug can be administered in an effective amount to the host using any suitable approach which achieves the desired therapeutic result. The amount and timing of active compound administered will, of course, be dependent on the host being treated, the instructions of the supervising medical specialist, on the time course of the exposure, on the manner of administration, on the pharmacokinetic properties of the particular active compound, and on the judgment of the prescribing physician. Thus, because of host to host variability, the dosages given below are a guideline and the physician can titrate doses of the compound to achieve the treatment that the physician considers appropriate for the host. In considering the degree of treatment desired, the physician can balance a variety of factors such as age and weight of the host, presence of preexisting disease, as well as presence of other diseases. Pharmaceutical formulations can be prepared for any desired route of administration including, but not limited to, oral, intravenous, or aerosol administration, as discussed in greater detail below.

The therapeutically effective dosage of any active compound described herein will be determined by the health care practitioner depending on the condition, size and age of the patient as well as the route of delivery. In one non-limited embodiment, a dosage from about 0.1 to about 200 mg/kg has therapeutic efficacy, with all weights being calculated based upon the weight of the active compound, including the cases where a salt is employed. In some embodiments, the dosage can be the amount of compound needed to provide a serum concentration of the active compound of up to between about 1 and 5, 10, 20, 30, or 40 μM. In some embodiments, a dosage from about 10 mg/kg to about 50 mg/kg can be employed for oral administration. Typically, a dosage from about 0.5 mg/kg to 5 mg/kg can be employed for intramuscular injection. In some embodiments, dosages can be from about 1 μmol/kg to about 50 μmol/kg, or, optionally, between about 22 μmol/kg and about 33 μmol/kg of the compound for intravenous or oral administration. An oral dosage form can include any appropriate amount of active material, including for example from 5 mg to, 50, 100, 200, or 500 mg per tablet or other solid dosage form.

In accordance with the presently disclosed methods, pharmaceutically active compounds as described herein can be administered orally as a solid or as a liquid, or can be administered intramuscularly, intravenously, or by inhalation as a solution, suspension, or emulsion. In some embodiments, the compounds or salts also can be administered by inhalation, intravenously, or intramuscularly as a liposomal suspension. When administered through inhalation the active compound or salt can be in the form of a plurality of solid particles or droplets having any desired particle size, and for example, from about 0.01, 0.1 or 0.5 to about 5, 10, 20 or more microns, and optionally from about 1 to about 2 microns. Compounds as disclosed in the present invention have demonstrated good pharmacokinetic and pharmacodynamics properties, for instance when administered by the oral or intravenous routes.

The pharmaceutical formulations can comprise an active compound described herein or a pharmaceutically acceptable salt thereof, in any pharmaceutically acceptable carrier. If a solution is desired, water may be the carrier of choice for water-soluble compounds or salts. With respect to the water-soluble compounds or salts, an organic vehicle, such as glycerol, propylene glycol, polyethylene glycol, or mixtures thereof, can be suitable. In the latter instance, the organic vehicle can contain a substantial amount of water. The solution in either instance can then be sterilized in a suitable manner known to those in the art, and for illustration by filtration through a 0.22-micron filter. Subsequent to sterilization, the solution can be dispensed into appropriate receptacles, such as depyrogenated glass vials. The dispensing is optionally done by an aseptic method. Sterilized closures can then be placed on the vials and, if desired, the vial contents can be lyophilized.

In addition to the active compounds or their salts, the pharmaceutical formulations can contain other additives, such as pH-adjusting additives. In particular, useful pH-adjusting agents include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate. Further, the formulations can contain antimicrobial preservatives. Useful antimicrobial preservatives include methylparaben, propylparaben, and benzyl alcohol. An antimicrobial preservative is typically employed when the formulations is placed in a vial designed for multi-dose use. The pharmaceutical formulations described herein can be lyophilized using techniques well known in the art.

For oral administration a pharmaceutical composition can take the form of solutions, suspensions, tablets, pills, capsules, powders, and the like. Tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate may be employed along with various disintegrants such as starch (e.g., potato or tapioca starch) and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate, and talc are often very useful for tableting purposes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules. Materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the compounds of the presently disclosed host matter can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents and/or suspending agents, as well as such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.

In yet another embodiment of the host matter described herein, there are provided injectable, stable, sterile formulations comprising an active compound as described herein, or a salt thereof, in a unit dosage form in a sealed container. The compound or salt is provided in the form of a lyophilizate, which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form liquid formulation suitable for injection thereof into a host. When the compound or salt is substantially water-insoluble, a sufficient amount of emulsifying agent, which is physiologically acceptable, can be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier. Particularly useful emulsifying agents include phosphatidyl cholines and lecithin.

Additional embodiments provided herein include liposomal formulations of the active compounds disclosed herein. The technology for forming liposomal suspensions is well known in the art. When the compound is an aqueous-soluble salt, using conventional liposome technology, the same can be incorporated into lipid vesicles. In such an instance, due to the water solubility of the active compound, the active compound can be substantially entrained within the hydrophilic center or core of the liposomes. The lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free. When the active compound of interest is water-insoluble, again employing conventional liposome formation technology, the salt can be substantially entrained within the hydrophobic lipid bilayer that forms the structure of the liposome. In either instance, the liposomes that are produced can be reduced in size, as through the use of standard sonication and homogenization techniques. The liposomal formulations comprising the active compounds disclosed herein can be lyophilized to produce a lyophilizate, which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.

Pharmaceutical formulations also are provided which are suitable for administration as an aerosol by inhalation. These formulations comprise a solution or suspension of a desired compound described herein or a salt thereof, or a plurality of solid particles of the compound or salt. The desired formulations can be placed in a small chamber and nebulized. Nebulization can be accomplished by compressed air or by ultrasonic energy to form a plurality of liquid droplets or solid particles comprising the compounds or salts. The liquid droplets or solid particles may for example have a particle size in the range of about 0.5 to about 10 microns, and optionally from about 0.5 to about 5 microns. In one embodiment, the solid particles provide for controlled release through the use of a degradable polymer. The solid particles can be obtained by processing the solid compound or a salt thereof, in any appropriate manner known in the art, such as by micronization. Optionally, the size of the solid particles or droplets can be from about 1 to about 2 microns. In this respect, commercial nebulizers are available to achieve this purpose. The compounds can be administered via an aerosol suspension of respirable particles in a manner set forth in U.S. Pat. No. 5,628,984, the disclosure of which is incorporated herein by reference in its entirety.

Pharmaceutical formulations also are provided which provide a controlled release of a compound described herein, including through the use of a degradable polymer, as known in the art.

When the pharmaceutical formulations suitable for administration as an aerosol is in the form of a liquid, the formulations can comprise a water-soluble active compound in a carrier that comprises water. A surfactant can be present, which lowers the surface tension of the formulations sufficiently to result in the formation of droplets within the desired size range when hosted to nebulization.

The term “pharmaceutically acceptable salts” as used herein refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with hosts (e.g., human hosts) without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the presently disclosed host matter.

Thus, the term “salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of the presently disclosed compounds. These salts can be prepared during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Basic compounds are capable of forming a wide variety of different salts with various inorganic and organic acids. Acid addition salts of the basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner. The free base form can be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner. The free base forms may differ from their respective salt forms in certain physical properties such as solubility in polar solvents. Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metal hydroxides, or of organic amines. Examples of metals used as cations, include, but are not limited to, sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines include, but are not limited to, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, and procaine. The base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid forms may differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents.

Salts can be prepared from inorganic acids sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic, phosphorus, and the like. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, laurylsulphonate and isethionate salts, and the like. Salts can also be prepared from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. and the like. Representative salts include acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like. Pharmaceutically acceptable salts can include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Also contemplated are the salts of amino acids such as arginate, gluconate, galacturonate, and the like. See, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19, which is incorporated herein by reference.

Preparation of Active Compounds

Syntheses

The disclosed compounds can be made by the following general schemes.

A method for the preparation of substituted tricyclic lactams is provided that includes efficient methods for the preparation of a tricyclic lactam ring system and subsequent displacement of an aryl sulfone with an amine.

In Scheme 1, diethyl succinate is employed to prepare the pyrimidine ester, 2, according to the method of A. Haidle, See, WO 2009/152027 entitled “5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives for MARK inhibition.” The ester intermediate 2 can be reduced by directly reacting the ester with a reducing agent such as lithium borohydride in a protic organic solvent such as ethanol to produce the corresponding primary alcohol. The primary alcohol can be reacted with a reagent such as phosphorus tribromide in an organic solvent such as dimethylforamide to produce the primary bromide 3. The primary bromide 3 can be condensed with the lactam 4 optionally at low temperature using a base such as lithium diisopropylamide in an organic solvent such as tetrahydrofuran to produce the lactam 5. Lactam 5 can be deprotected by directly reacting Compound 5 with an aqueous acid such as HCl=pH 1 solution. Lactam 6 can be directly reacted with an organic base such as 1,8-diazabicyclo[5.4.0]undec-7-ene in a protic solvent such as ethanol optionally at high temperature to cyclize Compound 5 to form the tricyclic lactam 7. The thiol moiety can be subsequently oxidized to the sulfone 8 by directly reacting Compound 7 with an oxidizing reagent such as meta-chloroperoxybenzoic acid. The sulfone, 8, can be directly reacted with an amine, 9, in the presence of a strong base such as lithium hexamethyldisilazane to form the tricyclic lactam 10.

In Scheme 2, the tricyclic lactam 7 is directly reacted with an oxidizing reagent such as 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to form the alkene 11. Alkene 11 can be directly reacted with an oxidizing reagent such as meta-chloroperoxybenzoic acid to form the sulfone intermediate 12. The sulfone, 12, can be condensed with an amine, 13, in the presence of a strong base such as lithium hexamethyldisilazane to form the tricyclic lactam 14.

Scheme 3 illustrates the synthesis of a di-protected lactam useful in the preparation of tricyclic lactams. Compound 15 is prepared according to the method of Arigon, J., See, US 2013/0289031 entitled “Pyrimidinone derivatives, preparation thereof and pharmaceutical use thereof” Compound 15 is protected with a suitable protecting group by directly reacting Compound 15 with di-tert-butyl carbonate (Boc anhydride) in the presence of an organic base such as triethylamine or diisopropylethylamine in an organic solvent such as dichloromethane to form the protected amine 16. The protected amine 16 can be directly reacted with methyl chloroacetate in the presence of a base such as potassium carbonate in an organic solvent such as acetonitrile to form the ester 17. The ester 17 can be cyclized by directly reacting the ester with an acid such as hydrochloric acid in a protic solvent such as methanol optionally at a high temperature to form the spirolactam 18. The lactam 18 can be directly reacted with a protecting reagent such as chloromethyl methyl ether (MOM-Cl) in the presence of an organic base such as diisopropylethylamine in an organic solvent such as dichloromethane optionally at a low or at ambient temperature to form the MOM-protected amine 19. The lactam 19 can be protected by directly reacting the lactam with a suitable protecting reagent such as chloromethyl methyl ether (MOM-Cl) in the presence of a base such as sodium bis(trimethylsilyl)amide in an organic solvent such as tetrahydrofuran optionally at a low temperature. Additional lactam intermediates such as Compounds 25 and 31 can be synthesized using analogous chemistry as described for the synthesis of Compound 4. The chemistry for the production of Compounds 25 and 31 is illustrated in Schemes 5 and 6.

Scheme 4 illustrates the coupling of a tricyclic lactam sulfone with an amine to generate compounds of Formula I, II, III, and IV.

Scheme 7 illustrates the preparation of the tricyclic lactam compound 33. Compound 32 is prepared according to the method of Tavares, See, U.S. Pat. No. 8,598,186. Compound 32 is directly reacted with sulfone 8 optionally in the presence of an organic base such as lithium hexamethyldisilazane to form the amine 33. The same chemistry can be employed to produce the alkene compound 34.

In one embodiment a lactam intermediate is treated with BOC-anhydride in the presence of an organic base such as triethylamine in an organic solvent such as dichloromethane. The Boc protected lactam is treated with carbon dioxide in the presence of a nickel catalyst to generate a carboxylic acid. The carboxylic acid is reacted with thionyl chloride in the presence of an organic solvent such as toluene. The resulting acid chloride is treated with an amine to generate an amide that can be deprotected with a strong acid such as trifluoroacetic acid to generate the final target compound.

Alternatively, the lactam can be generated by reacting the carboxylic acid with a protected amine in the presence of a strong acid and a dehydrating agent, which can be together in one moiety as a strong acid anhydride. Examples of strong acid anhydrides include, but are not limited to, trifluoroacetic acid anhydride, tribromoacetic acid anhydride, trichloroacetic acid anhydride, or mixed anhydrides. The dehydrating agent can be a carbodiimide based compound such as but not limited to DCC (N,N-dicyclohexylcarbodiimide), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or DIC (N,N-diisopropylcarbodiimide). An additional step may be necessary to take off the N-protecting group and the methodologies are known to those skilled in the art

Alternatively, the SMe moiety bonded to the pyrimidine ring can be substituted with any leaving group that can be displaced by a primary amine, for example to create an intermediate for a final product such as Br, I, F, SO₂Me, SOalkyl, SO₂alkyl. See, for Example, PCT/US2013/037878 to Tavares.

Other amine intermediates and final amine compounds can be synthesized by those skilled in the art. It will be appreciated that the chemistry can employ reagents that comprise reactive functionalities that can be protected and de-protected and will be known to those skilled in the art at the time of the invention. See for example, Greene, T. W. and Wuts, P. G. M., Greene's Protective Groups in Organic Synthesis, 4^(th) edition, John Wiley and Sons.

[4-Chloro-2-(methylthio)pyrimidin-5-yl]methanol

4-Chloro-2-methylsulfanyl-5-pyrimidinecarboxylate ethyl ester (62 g, 260 mmol) was dissolved in anhydrous tetrahydrofuran (500 mL) in a 3-necked 5 L round bottomed flask fitted with a mechanical stirrer, addition funnel, temperature probe and nitrogen inlet. The solution was cooled to 0° C. Diisobutylaluminum hydride in tetrahydrofuran (1M solution, 800 mL) was added dropwise over a period of 2 hours. After the addition was complete, the reaction mixture was kept at 0° C. for 0.5 hours. The reaction was quenched at 0° C. by the slow addition of saturated aqueous sodium sulfate (265.3 mL, 530.7 mmol) keeping the internal reaction temperature below 10° C. Ethyl acetate (900 mL) was added and the reaction slowly warmed to room temperature overnight. 6M HCl was added till the reaction mixture was slightly acidic (pH 6). The reaction mixture was filtered thru a pad of Celite® and the aluminum salts were washed with ethyl acetate (1 L). The filtrate was poured into a separatory funnel and washed twice with water (600 mL) and finally with brine (600 mL). The organic layer was dried over sodium sulfate, filtered thru Celite® and the solvent concentrated in vacuo to afford 39.2 g (77% crude yield) of a dark yellow oil. The material was used as is for the next step. NMR (CDCl₃) δ 8.56 (s, 1H), 4.76 (s, 2H), 2.59 (s, 3H); MS (ESI+) for C₆H₇ClN₂OS m/z 191.0 (M+H)⁺.

4-Chloro-2-(methylthio)pyrimidine-5-carbaldehyde

[4-Chloro-2-(methylthio)pyrimidin-5-yl]methanol (39.2 g, 206 mmol) was taken up in methylene chloride (520 mL) at room temperature. Manganese(IV) oxide (140 g, 1.60 mol) was added in one portion and the reaction mixture stirred at room temperature overnight. The reaction mixture was filtered through a pad of Celite® and washed with methylene chloride. The filtrate was concentrated under reduced pressure to afford a dark yellow semisolid. The crude product was purified by reverse phase chromatography running a gradient of 1:9 acetonitrile:water (0.1% TFA) to 100% acetonitrile (0.1% TFA). The desired fractions were combined and the acetonitrile was removed under reduced pressure causing precipitation of the desired product. The solids were removed by filtration and the solids washed with water and dried under vacuum at 50° C. Affords 16.6 g (43% yield) of the desired product as a white solid. NMR (CDCl₃) δ 10.32 (s, 1H), 8.88 (s, 1H), 2.65 (s, 3H); MS (ESI+) for C₆H₅ClN₂OS m/z 189.0 (M+H)⁺.

General Procedure A.

Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate

Isopropylmagnesium chloride:lithium chloride complex (4.43 g, 30.5 mmol, 25.4 mL of a 1.2M solution) was added to tetrahydrofuran (104 mL) in a 500 mL round bottomed flask which had been flame dried and cooled under Argon. The solution was cooled to −15° C. Ethyl propiolate (3.26 mL, 32.1 mmol) was added dropwise affording a yellow solution. Stirring was continued at −15° C. for 30 minutes and then 4-Chloro-2-(methylthio)pyrimidine-5-carbaldehyde (6.06 g, 32.1 mmol) in tetrahydrofuran (52 mL) was added rapidly. After 10 minutes, the reaction was quenched by the addition of saturated aqueous ammonium chloride (40 mL). The reaction mixture was warmed to room temperature and poured into a separatory funnel partitioning between ethyl acetate (200 mL) and water (100 mL). The organic layer removed and the aqueous layer extracted with ethyl acetate (100 mL). The combined organic layers were washed with brine (100 mL), dried over sodium sulfate, filtered and the solvent removed in vacuo to afford a dark red oil. The product was purified by silica gel chromatography using a gradient of 1:4 to 2:3 ethyl acetate:hexanes which afforded 3.76 g (37% yield) of the desired product as a light red oil. NMR (CDCl₃) δ 8.75 (s, 1H), 5.81 (d, 1H, J=6.0 Hz), 2.72 (bs, 1H), 2.60 (s, 3H), 1.33 (t, 3H, J=7.2 Hz); MS (ESI+) for C₁₁H₁₁ClN₂O₃S m/z 287.9 (M+H)⁺.

tert-Butyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate

Following General Procedure A and using tert-butyl propiolate affords the desired product in 74% yield as a viscous yellow oil. NMR (CDCl₃) δ 8.75 (s, 1H), 5.79 (d, 1H, J=5.4 Hz), 4.27 (q, 2H, J=7.2 Hz), 2.97 (d, 1H, J=5.4 Hz), 2.60 (s, 3H), 1.52 (s, 9H); MS (ESI+) for C₁₃H₁₅ClN₂O₃S m/z 314.9 (M+H)⁺.

General Procedure B.

Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate

Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate (3.40 g, 11.8 mmol) was taken up in 1,4-Dioxane (100 mL) at room temperature under argon. Triethylamine (3.3 mL, 24 mmol) was added and the mixture heated to 60° C. for 1 h. The reaction mixture was cooled to room temperature and the solvent removed in vacuo. The resultant dark orange oil was re-evaporated twice with toluene. Affords the desired product (3:1 E:Z double bond isomers) in 99% yield as a dark orange oil. NMR (CDCl₃) (major E isomer) δ 8.69 (s, 1H), 7.65 (d, 1H, J=18.0 Hz), 6.81 (d, 1H, J=18.0 Hz), 4.32 (q, 2H, J=6.0 Hz), 2.64 (s, 3H), 1.36 (t, 3H, J=6.0 Hz); NMR (CDCl₃) (minor Z isomer) δ 8.87 (s, 1H), 6.89 (d, 1H, J=12.0 Hz), 6.23 (d, 1H, J=12.0 Hz), 4.14 (q, 2H, J=6.0 Hz), 2.63 (s, 3H), 1.23 (t, 3H, J=6.0 Hz); MS (ESI+) for C₁₁H₁₁ClN₂O₃S m/z 287.0 (M+H)⁺.

tert-Butyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate

Isomerization of tert-butyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate using General Procedure B affords the desired product (5:1 E:Z double bond isomers) in a 99% yield as a viscous dark yellow oil. NMR (CDCl₃) (major E isomer) δ 8.67 (s, 1H), 7.54 (d, 1H, J=15.6 Hz), 6.72 (d, 1H, J=15.6 Hz), 2.64 (s, 3H), 1.54 (s, 9H); NMR (CDCl₃) (minor Z isomer) δ 8.86 (s, 1H), 6.76 (d, 1H, J=12.0 Hz), 6.18 (d, 1H, J=12.0 Hz), 2.63 (s, 3H), 1.40 (s, 9H); MS (ESI+) for C₁₃H₁₅ClN₂O₃S m/z 314.9 (M+H)⁺.

General Procedure C.

Ethyl 2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate (3.40 g, 11.8 mmol) was taken up in acetonitrile (20 mL) at room temperature. 1-{[(Triisopropylsilyl)oxy]methyl}cyclopentanamine (3.86 g, 14.2 mmol) was added followed by triethylamine (3.30 mL, 23.7 mmol). The mixture was stirred at room temperature overnight. The reaction mixture was transferred to a separatory funnel transferring with ethyl acetate (250 mL). The organic layer was washed twice with a 10% citric acid (aq) (20 mL)/brine (60 mL) mixture. The organic layer was dried over sodium sulfate, filtered and the solvent removed in vacuo to afford a yellow oil. The product was purified by silica gel chromatography using a gradient from 1:9 to 2:3 ethyl acetate:hexanes which afforded 2.48 g (41% yield) of the desired product as a pale yellow oil. NMR (CDCl₃) δ 8.58 (s, 1H), 4.72 (m, 1H), 4.46 (m, 1H), 4.16 (q, 2H, J=6.9 Hz), 3.52 (m, 1H), 2.96 (m, 2H), 2.54 (s, 3H), 2.31 (m, 3H), 1.81-1.52 (m, 5H), 1.24 (t, 3H, J=6.9 Hz) 1.08-0.96 (m, 21H); MS (ESI+) for C₂₆H₄₃N₃O₄SSi m/z 522.2 (M+H)⁺.

Ethyl 2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Cyclization of ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and 1-{[(triisopropylsilyl)oxy]methyl}cyclohexanamine using General Procedure C afforded the desired product in 17% yield as a pale yellow oil. NMR (CDCl₃) δ 8.63 (s, 1H), 4.83 (m, 1H), 4.73 (m, 1H), 4.14 (q, 2H, J=6.0 Hz), 3.86 (m, 1H), 2.97 (m, 2H), 2.55 (s, 3H), 1.97 (m, 1H), 1.72-1.48 (m, 9H), 1.23 (t, 3H, J=6.0 Hz), 1.13-0.95 (m, 21H); MS (ESI+) for C₂₇H₄₅N₃O₄SSi m/z 536.2 (M+H)⁺.

Ethyl 8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Cyclization of ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and 1-{[tert-butyl(dimethyl)silyl]oxy}-2-methylpropan-2-amine using General Procedure C afforded the desired product in 52% yield as a pale yellow oil. NMR (CDCl₃) δ 8.62 (s, 1H), 4.94 (m, 1H), 4.18 (m, 3H), 3.63 (m, 1H), 2.93 (m, 2H), 2.57 (s, 3H), 1.71 (s, 3H), 1.55 (s, 3H), 1.23 (t, 3H, J=7.2 Hz), 0.89 (s, 9H), 0.06 (s, 3H), 0.01 (s, 3H); MS (ESI+) for C₂₁H₃₅N₃O₄SSi m/z 454.3 (M+H)⁺.

Ethyl 2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Cyclization of ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and 2-[(triisopropylsilyl)oxy]ethanamine using General Procedure C afforded the desired product in 45% yield as a pale yellow oil. NMR (CDCl₃) δ 8.60 (s, 1H), 4.72 (m, 1H), 4.63 (m, 1H), 4.19 (q, 2H, J=6.0 Hz), 3.97 (m, 2H), 3.22 (m, 1H), 3.01 (m, 2H), 2.54 (s, 3H), 1.28 (t, 3H, J=6.0 Hz), 1.17-1.02 (m, 21H); MS (ESI+) for C₂₂H₃₇N₃O₄SSi m/z 468.1 (M+H)⁺.

tert-Butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Cyclization of tert-Butyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and 0.5 M ammonia/dioxane using General Procedure C afforded the desired product in 60% yield as an off-white solid. NMR (CDCl₃) δ 8.66 (s, 1H), 6.18 (bs, 1H), 4.34 (m, 1H), 3.05-2.80 (m, 2H), 2.56 (s, 3H), 1.51 (s, 9H); MS (ESI+) for C₁₃H₁₇N₃O₃S m/z 296.0 (M+H)⁺.

General Procedure D.

2-(Methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid

Ethyl 2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate (2.48 g, 4.75 mmol) was taken up in tetrahydrofuran (10 mL) and acetonitrile (10 mL) at room temperature. 1M Sodium hydroxide (10 mL, 10 mmol) was added at room temperature for 1 hour. The reaction was quenched by the addition of 10% citric acid till pH ca 6-7. The reaction mixture was transferred to a separatory funnel with water (30 mL) and ethyl acetate (150 mL). The aqueous layer was removed and the organic layer washed with brine (50 mL). The organic layer was dried over sodium sulfate, filtered and the solvent concentrated in vacuo to afford the 2.08 g (89% yield) of the desired product as a dark yellow oil. NMR (CDCl₃) δ 8.46 (s, 1H), 4.58 (m, 1H), 4.39 (m, 1H), 3.71 (m, 1H), 2.88 (m, 2H), 2.51 (s, 3H), 2.26 (m, 3H), 1.97-1.45 (m, 6H) 1.12-0.92 (m, 21H); MS (ESI+) for C₂₄H₃₉N₃O₄SSi m/z 494.2 (M+H)⁺.

2-(Methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid

Saponification of ethyl 2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate using General Procedure D affords the desired product in 95% yield as a dark yellow oil. NMR (CDCl₃) δ 8.58 (s, 1H), 4.75 (m, 1H), 4.53 (m, 1H), 3.96 (m, 1H), 2.99 (m, 2H), 2.54 (s, 3H), 1.93-1.48 (m, 10H), 1.13-0.95 (m, 21H); MS (ESI+) for C₂₅H₄₁N₃O₄SSi m/z 508.1 (M+H)⁺.

8-(2-{[tert-Butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid

Saponification of ethyl 8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate using General Procedure D affords the desired product in 99% yield as a pale yellow foam. NMR (CDCl₃) δ 8.69 (s, 1H), 4.88 (m, 1H), 4.51 (m, 1H), 3.82 (m, 1H), 3.17 (m, 1H), 2.79 (m, 1H), 2.57 (s, 3H), 1.65 (s, 3H), 1.60 (s, 3H), 0.92 (s, 9H), 0.11 (s, 6H); MS (ESI+) for C₁₉H₃₁N₃O₄SSi m/z 426.3 (M+H)⁺.

2-(Methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid

Saponification of ethyl 2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate using General Procedure D affords the desired product in 98% yield as an orange foam. NMR (CDCl₃) δ 8.56 (s, 1H), 4.71 (m, 1H), 4.54 (m, 1H), 3.99 (m, 2H), 3.32 (m, 1H), 3.01 (m, 2H), 2.53 (s, 3H), 1.16-0.98 (m, 21H); MS (ESI+) for C₂₀H₃₃N₃O₄SSi m/z 440.2 (M+H)⁺.

2-(Methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid

tert-Butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate (220 mg, 0.74 mmol) was taken up in trifluoroacetic acid (5 mL) at room temperature under argon. The mixture was stirred at room temperature for 45 minutes. The solvent was removed in vacuo to a pink oil which was re-evaporated first from toluene and finally methanol affording 180 mg (99% yield) of the desired product as an off-white solid. NMR (MeOH-d₄) δ 8.49 (s, 1H), 4.59 (m, 1H), 3.16-2.91 (m, 2H), 2.63 (s, 3H); MS (ESI+) for C₉H₉N₃O₃S m/z 240.0 (M+H)⁺.

General Procedure E.

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

2-(Methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid (2.08 g, 4.21 mmol) was taken up in N,N-dimethylformamide (30 mL) at room temperature. 1-(2,4-dimethoxyphenyl)methanamine (1.26 mL, 8.42 mmol) was added followed by the addition of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (4.80 g, 12.6 mmol) and N,N-diisopropylethylamine (4.40 mL, 25.3 mmol). The reaction mixture was stirred overnight at room temperature. The product was diluted with water (50 mL) and poured into a separatory funnel. The mixture was extracted with twice with ethyl acetate (150 mL) and the combined organic layers were thrice washed with half-saturated aqueous LiCl (20 mL). The combined organic layers were dried over sodium sulfate, filtered and the solvent removed in vacuo to afford a dark yellow oil. The product was purified by silica gel chromatography using a gradient from 1:4 to 2:3 ethyl acetate:hexanes which afforded 2.39 g (88% yield) of the desired product as a brown sticky solid. NMR (CDCl₃) δ 8.58 (s, 1H), 7.07 (m, 1H), 6.62 (m, 1H), 6.39 (m, 2H), 4.56 (m, 1H), 4.38 (m, 2H), 4.20 (m, 1H), 3.80 (s, 3H), 3.66 (s, 3H), 3.48 (m, 1H), 3.02 (m, 2H), 2.55 (s, 3H), 2.35 (m, 2H), 2.06 (m, 1H), 1.70-1.31 (m, 5H), 1.09-0.90 (m, 21H); MS (ESI+) for C₃₃H₅₀N₄O₅SSi m/z 643.2 (M+H)⁺.

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Coupling reaction of 2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid using General Procedure E afforded the desired product in 52% yield as a yellow solid. NMR (CDCl₃) δ 8.60 (s, 1H), 6.87 (m, 2H), 6.37 (m, 2H), 4.71 (m, 1H), 4.49-4.14 (m, 4H), 3.79 (s, 3H), 3.70 (s, 3H), 3.18 (m, 1H), 2.86 (m, 1H), 2.65 (m, 1H), 2.54 (s, 3H), 2.20 (m, 1H), 1.98 (m, 2H), 1.61-1.48 (m, 6H), 1.08-0.92 (bs, 21H); MS (ESI+) for C₃₄H₅₂N₄O₅SSi m/z 657.2 (M+H)⁺.

8-(2-{[tert-Butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-N-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Coupling reaction of 8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid using General Procedure E afforded the desired product in 86% yield as a viscous yellow oil. NMR (CDCl₃) δ 8.58 (s, 1H), 6.95 (m, 1H), 6.80 (m, 1H), 6.37 (m, 2H), 4.71 (m, 1H), 4.27 (m, 2H), 4.13 (m, 1H), 3.84 (m, 1H), 3.79 (s, 3H), 3.70 (s, 3H), 3.15 (m, 1H), 2.77 (m, 1H), 2.56 (s, 3H), 1.64 (s, 3H), 1.58 (s, 3H), 0.85 (s, 9H), 0.02 (s, 3H), -0.03 (s, 3H); MS (ESI+) for C₂₈H₄₂N₄O₅SSi m/z 575.4 (M+H)⁺.

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Coupling reaction of 2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid using General Procedure E afforded the desired product in 57% yield as a dark yellow solid. NMR (CDCl₃) δ 8.59 (s, 1H), 7.06 (m, 1H), 6.39 (m, 3H), 4.51 (m, 2H), 4.32 (m, 2H), 3.93 (m, 2H), 3.81 (s, 3H), 3.73 (s, 3H), 3.18 (m, 1H), 3.01 (m, 2H), 2.54 (s, 3H), 1.11-0.95 (m, 21H); MS (ESI+) for C₂₉H₄₄N₄O₅SSi m/z 589.4 (M+H)⁺.

2-(Methylthio)-5-oxo-N-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Coupling reaction of 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid using General Procedure E afforded the desired product in 94% yield as a dark yellow oil. NMR (CDCl3) δ 8.64 (s, 1H), 6.09 (bs, 1H), 6.04 (bs, 1H), 4.25 (m, 1H), 3.68 (m, 2H), 2.86 (m, 2H), 2.54 (s, 3H), 2.07-1.54 (m, 8H), 1.33-0.96 (m, 21H); MS (ESI+) for C₂₄H₄₀N₄O₃SSi m/z 493.1 (M+H)⁺.

General Procedure F.

N-(2,4-Dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclopentyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide (2.39 g, 3.72 mmol) was taken up in tetrahydrofuran (42 mL) at room temperature. Tetra-n-butylammonium fluoride (5.6 mL, 5.6 mmol, 1M solution in THF) was added and the reaction stirred for 10 minutes at room temperature. The reaction mixture was concentrated in vacuo to an orange oil and was transferred to a separatory funnel and partitioned between ethyl acetate (200 mL) and water (50 mL). The aqueous layer was removed and the organic layer washed with water (50 mL) and brine (50 mL). The organic layer was dried over sodium sulfate, filtered and the solvent removed in vacuo to afford a dark yellow semi-solid. The product was purified by reverse phase chromatography using a gradient from 1:9 to 3:2 acetonitrile:water (0.1% TFA). Lyophilization of the desired fractions afforded 1.81 g (99% yield) of the desired product as a dark yellow powder. NMR (CDCl₃) δ 8.59 (s, 1H), 7.21 (m, 1H), 7.02 (m, 1H), 6.39 (m 2H), 4.56 (m, 1H), 4.29 (m, 2H), 3.79 (s, 3H), 3.75 (s, 3H), 3.70 (m, 2H), 3.41 (m, 1H), 3.20 (m, 1H), 2.87 (m, 1H), 2.55 (s, 3H), 2.18 (m, 1H), 1.97-1.59 (m, 7H); MS (ESI+) for C₂₄H₃₀N₄O₅S m/z 487.1 (M+H)⁺.

N-(2,4-Dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclohexyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation of N-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide using General Procedure F afforded the desired product in 97% yield as a yellow powder. NMR (CDCl₃) δ 8.69 (s, 1H), 7.58 (m, 1H), 7.07 (m, 1H), 6.48 (m, 2H), 4.72 (m, 2H), 4.38 (m, 2H), 3.89 (s, 3H), 3.87 (s, 3H), 3.31 (m, 1H), 2.99 (m, 1H), 2.81 (m, 1H), 2.64 (s, 3H), 2.11 (m, 3H), 1.94-1.58 (m, 7H); MS (ESI+) for C₂₅H₃₂N₄O₅S m/z 501.1 (M+H)⁺.

8-(2-Hydroxy-1,1-dimethylethyl)-N-(4-methoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation of 8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-N-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide using General Procedure F afforded the desired product in 98% yield as a yellow powder. NMR (CDCl₃) δ 8.61 (s, 1H), 7.51 (m, 1H), 7.01 (m, 1H), 6.37 (m, 2H), 4.72 (m, 1H), 4.65 (m, 1H), 4.26 (m, 2H), 3.80 (s, 3H), 3.76 (s, 3H), 3.63 (m, 1H), 3.18 (m, 1H), 2.80 (m, 1H), 2.57 (s, 3H), 1.58 (s, 3H), 1.56 (s, 3H); MS (ESI+) for C₂₂H₂₈N₄O₅S m/z 461.4 (M+H)⁺.

N-(2,4-Dimethoxybenzyl)-8-(2-hydroxyethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation of N-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide using General Procedure F afforded the desired product in 97% yield as a pale yellow powder. NMR (CDCl₃) δ 8.59 (s, 1H), 7.02 (m, 1H), 6.69 (m, 1H), 6.43 (m, 2H), 4.34 (m, 3H), 3.88 (m, 4H), 3.81 (s, 3H), 3.78 (s, 3H), 3.02 (m, 2H), 2.55 (s, 3H); MS (ESI+) for C₂₀H₂₄N₄O₅S m/z 432.9 (M+H)⁺.

N-[1-(Hydroxymethyl)cyclopentyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation of 2-(methylthio)-5-oxo-N-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide using General Procedure F afforded the desired product in 31% yield as an off white powder. NMR (CDCl₃) δ 8.66 (s, 1H), 6.19 (bs, 1H), 5.97 (bs, 1H), 4.31 (m, 1H), 3.69 (s, 2H), 2.92 (m, 2H), 2.56 (s, 3H), 1.95-1.65 (m, 9H); MS (ESI+) for C₁₅H₂₀N₄O₃S m/z 337.0 (M+H)⁺.

General Procedure G.

8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

N-(2,4-Dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclopentyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide (1.81 g, 3.72 mmol) was taken up in methylene chloride (30 mL) at room temperature. Triethylamine (1.3 mL, 9.3 mmol) was added followed by the rapid addition of methanesulfonyl chloride (0.43 mL, 5.6 mmol). The reaction mixture was stirred at room temperature for 30 minutes before being heated at reflux overnight. The solvent was removed in vacuo affording a brown semi-solid. The product was purified by reverse phase chromatography using a gradient from 1:9 to 3:2 acetonitrile:water (0.1% TFA). Lyophilization of the desired fractions gave 764 mg (44% yield) of the desired product as a light brown powder. NMR (CDCl₃) δ 8.49 (s, 1H), 7.13 (m, 1H), 6.43 (m, 2H), 5.49 (m, 1H), 4.37-4.16 (m, 4H), 3.81 (s, 3H), 3.80 (s, 3H), 3.29 (m, 1H), 2.95 (m, 1H), 2.82 (s, 3H), 2.41 (m, 1H), 1.99-1.52 (m, 7H); MS (ESI+) for C₂₄H₂₈N₄O₄S m/z 469.1 (M+H)⁺.

8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

Mesylation and cyclization of N-(2,4-dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclohexyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide using General Procedure G afforded the desired product in 73% yield as a white powder. NMR (MeOH-d₄) δ 8.48 (s, 1H), 7.14 (m, 1H), 6.43 (m, 1H), 6.39 (m, 1H), 5.61 (m, 1H), 4.31 (m, 1H), 4.21 (m, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.38 (m, 1H), 3.22 (m, 1H), 2.95 (m, 1H), 2.82 (s, 3H), 2.22 (m, 1H), 1.99-1.09 (m, 9H); MS (ESI+) for C₂₅H₃₀N₄O₄S m/z 483.1 (M+H)⁺.

8-(2,4-Dimethoxybenzyl)-10,10-dimethyl-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

Mesylation and cyclization of 8-(2-Hydroxy-1,1-dimethylethyl)-N-(4-methoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide using General Procedure G afforded the desired product in 82% yield as a yellow powder. NMR (CDCl₃) δ 8.45 (s, 1H), 7.14 (m, 1H), 6.40 (m, 2H), 5.56 (m, 1H), 4.40 (m, 1H), 4.31 (m, 2H), 4.13 (m, 1H), 3.81 (s, 3H), 3.79 (s, 3H), 3.28 (m, 1H), 2.88 (m, 1H), 2.79 (s, 3H), 1.80 (s, 3H), 1.45 (s, 3H); MS (ESI+) for C₂₂H₂₆N₄O₄S m/z 443.5 (M+H)⁺.

8-(2,4-Dimethoxybenzyl)-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

Mesylation and cyclization of N-(2,4-dimethoxybenzyl)-8-(2-hydroxyethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide using General Procedure G afforded the desired product in 81% yield as a light brown powder. NMR (CDCl₃) δ 8.52 (s, 1H), 7.11 (m, 1H), 6.44 (m, 2H), 5.31 (m, 1H), 4.68-4.29 (m, 5H), 4.04 (m, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.20 (m, 1H), 3.01 (m, 1H), 2.82 (s, 3H); MS (ESI+) for C₂₀H₂₂N₄O₄S m/z 415.0 (M+H)⁺.

General Procedure H.

2′-(Methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione (764 mg, 1.63 mmol) was taken up in trifluoroacetic acid (10 mL) at room temperature under argon. The mixture was heated to 75° C. for 6 hours, cooled to room temperature and left to stir overnight. The solvent was removed in vacuo to afford a purple oil. The product was purified by reverse phase chromatography using a gradient from 100% water (0.1% TFA) to 1:1 acetonitrile:water (0.1% TFA). Lyophilization of the desired fractions afforded 117 mg (23% yield) of the desired product as a pale yellow powder. NMR (CDCl₃) δ 8.52 (s, 1H), 5.58 (m, 2H), 4.34 (bs 2H), 3.29 (m, 1H), 3.06 (m, 1H), 2.84 (s, 3H), 2.48 (m, 1H), 2.31 (m, 1H), 2.11-1.65 (m, 6H); MS (ESI+) for C₁₅H₁₈N₄O₂S m/z 319.0 (M+H)⁺.

2′-(Methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

Removal of the dimethoxybenzyl group of 8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione using General Procedure H afforded the desired product in 23% yield as a white powder. NMR (MeOH-d₄) δ 8.60 (s, 1H), 4.78 (m, 1H), 4.54 (m, 2H), 3.38 (m, 1H), 2.86 (s, 3H), 2.84 (m, 1H), 2.12-1.30 (m, 10H); MS (ESI+) for C₁₆H₂₀N₄O₂S m/z 333.1 (M+H)⁺.

10,10-Dimethyl-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

Removal of the dimethoxybenzyl group of 8-(2,4-dimethoxybenzyl)-10,10-dimethyl-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione using General Procedure H afforded the desired product in 56% yield as a white powder. NMR (CDCl₃) δ 8.54 (s, 1H), 5.65 (m, 1H), 5.51 (bs, 1H), 4.31 (s, 2H), 3.23 (m, 1H), 3.04 (m, 1H), 2.85 (s, 3H), 1.78 (s, 3H), 1.68 (s, 3H); MS (ESI+) for C₁₃H₁₆N₄O₂S m/z 293.2 (M+H)⁺.

2-(Methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

Removal of the dimethoxybenzyl group of 8-(2,4-dimethoxybenzyl)-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione using General Procedure H afforded the desired product in 21% yield as a brown powder. NMR (MeOH-d₄) δ 8.65 (s, 1H), 4.78-4.05 (m, 5H), 3.32 (m, 2H), 3.01 (m, 1H), 2.87 (s, 3H); MS (ESI+) for C₁₁H₁₂N₄O₂ m/z 265.0 (M+H)⁺.

General Procedure I.

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

2′-(Methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione (117 mg, 0.367 mmol) was taken up in N,N-dimethylacetamide (4.0 mL, 43 mmol) at room temperature under argon. 5-(4-methylpiperazin-1-yl)pyridin-2-amine (100 mg, 0.55 mmol) was added and the reaction mixture was heated just to 150° C. and then immediately removed from the heat and cooled to room temperature. The product was purified by reverse phase chromatography using a gradient from 100% Water (0.1% TFA) to 1:1 acetonitrile:water (0.1% TFA). Lyophilization of the desired fractions afforded 11 mg (7% yield) of the desired product as an orange powder. NMR (MeOH-d₄) δ 8.54 (s, 1H); 8.06 (m, 1H), 7.85 (m, 1H), 7.69 (m, 1H), 4.69 (m, 1H), 4.39 (m, 2H), 3.95 (m, 2H), 3.69 (m, 2H), 3.39-3.15 (m, 5H), 3.02 (s, 3H), 2.77 (m, 1H), 2.21 (m, 1H), 2.09-1.67 (m, 7H); MS (ESI+) for C₂₄H₃₀N₈O₂ m/z 463.1 (M+H)⁺.

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

S_(N)Ar reaction using General Procedure I and 2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione afforded the desired product in 38% yield as a yellow powder. NMR (MeOH-d₄) δ 8.52 (s, 1H), 8.07 (m, 1H), 7.89 (bs, 1H), 7.71 (m, 1H), 4.71 (m, 1H), 4.40 (m, 2H), 3.94 (m, 2H), 3.68 (m, 2H), 3.35-3.22 (m, 5H), 3.02 (s, 3H), 2.73 (m, 1H), 2.02-1.25 (m, 10H); MS (ESI+) for C₂₅H₃₂N₈O₂ m/z 477.2 (M+H)⁺.

10,10-Dimethyl-2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

S_(N)Ar reaction using General Procedure I and 10,10-dimethyl-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione afforded the desired product in 10% yield as a yellow powder. NMR (MeOH-d₄) δ 8.52 (s, 1H), 8.05 (m, 2H), 7.86 (m, 1H), 7.67 (m, 1H), 4.66 (m, 1H), 4.33 (m, 2H), 3.93 (m, 2H), 3.68 (m, 2H), 3.38-3.21 (m, 5H), 3.01 (s, 3H), 2.72 (m, 1H), 1.65 (s, 3H), 1.54 (s, 3H); MS (ESI+) for C₂₂H₂₈N₈O₂ m/z 437.4 (M+H)⁺.

2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

S_(N)Ar reaction using General Procedure I and 2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione afforded the desired product in 15% yield as an orange powder. NMR (DMSO-d₆) δ 8.41 (s, 1H), 8.05 (m, 1H), 7.93 (m, 1H), 7.82 (m, 1H), 4.59-4.34 (m, 3H), 4.03-3.84 (m, 4H), 3.49 (m, 2H), 3.30-3.09 (m, 6H), 2.80 (s, 3H), 2.80-2.67 (m, 2H); MS (ESI+) for C₂₀H₂₄N₈O₂ m/z 409.1 (M+H)⁺.

General Procedure J.

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydro-7′H-dispiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5′,2″-[1,3]dithian]-7′-one

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione (90.0 mg, 0.189 mmol) and 1,3-propanedithiol (0.0379 mL, 0.378 mmol) were taken up in toluene (5 mL) at room temperature under argon. p-Toluenesulfonic acid (0.02 g, 0.1 mmol) was then added. The reaction vessel was fitted with a condenser and the reaction mixture heated at reflux overnight. The reaction mixture was cooled to room temperature and the solvent removed in vacuo affording a thick dark yellow oil. The product was purified by reverse phase chromatography using a gradient from 100% water (0.1% TFA) to 3:2 acetonitrile:water (0.1% TFA). Lyophilization of the desired fractions afforded 35 mg (33% yield) of the desired product as a pale yellow powder. NMR (MeOH-d₄) δ 8.52 (s, 1H), 7.90 (m, 1H), 7.84 (m, 1H), 7.52 (m, 1H), 4.64 (m, 1H), 4.53 (m, 1H), 4.16 (m, 1H), 3.60 (m, 2H), 3.41-3.26 (m, 6H), 3.01 (s, 3H), 2.91 (m, 1H), 2.75 (m, 1H), 2.61 (m, 1H), 2.21 (m, 1H), 2.11 (m, 1H), 1.95-1.72 (m, 10H), 1.61 (m, 1H), 1.33 (m 2H); MS (ESI+) for C₂₈H₃₈N₈OS₂ m/z 567.1 (M+H)⁺.

10′,10′-Dimethyl-2′-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,9′,10′-tetrahydrospiro[1,3-dithiane-2,5′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidin]-7′(8′H)-one

Dithiane formation using General Procedure J and 10,10-dimethyl-2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione afforded the desired product in 43% yield as an orange powder. NMR (MeOH-d₄) δ 8.53 (s, 1H), 7.88 (m, 1H), 7.82 (m, 1H), 7.47 (m, 1H), 4.47 (m, 1H), 4.41 (m, 1H), 4.16 (m, 1H), 3.92-3.15 (m, 11H), 3.00 (s, 3H), 2.90-2.81 (m, 3H), 2.21 (m, 1H), 1.87 (m, 1H), 1.60 (s, 3H), 1.48 (s, 3H); MS (ESI+) for C₂₅H₃₄N₈OS₂ m/z 527.1 (M+H)⁺.

General Procedure K.

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidin]-7′(5′H)-one

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′, 8′,9′-tetrahydro-7′H-dispiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5′,2″-[1,3]dithian]-7′-one (35 mg, 0.062 mmol) in ethanol (1 ml) was added to Raney nickel (1 mL of the aqueous slurry which was washed thrice with ethanol decanting off the ethanol after each washing) in ethanol (3 mL) under argon. The reaction mixture was heated to 45° C. for 30 minutes. After cooling to room temperature, the reaction mixture was filtered through a pad of Celite® washing with ethanol. The solvent was removed in vacuo affording a yellow oil. The product was purified by reverse phase chromatography using a gradient from 100% water (0.1% TFA) to 3:2 acetonitrile:water (0.1% TFA). Lyophilization of the desired fractions afforded 4 mg (14% yield) of the desired product as a pale yellow powder. NMR (CDCl₃) δ 7.95 (m, 1H), 7.93 (s, 1H), 7.77 (m, 1H), 7.51 (m, 1H), 4.57 (m, 1H), 4.51 (m, 1H), 4.35 (m, 1H), 3.89 (m, 2H), 3.69 (m, 2H), 3.37 (m, 2H), 3.15 (m, 2H), 3.01 (s, 3H), 2.79 (m, 1H), 2.58 (m, 1H), 2.39 (m, 1H), 2.05-1.45 (m, 11H); MS (ESI+) for C₂₅H₃₄N₈O m/z 463.1 (M+H)⁺.

10,10-Dimethyl-2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidin-7(8H)-one

Desulfurization using General Procedure K and 10′,10′-dimethyl-2′-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,9′,10′-tetrahydrospiro[1,3-dithiane-2,5′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidin]-7′(8′H)-one afforded the desired product in 11% yield as a yellow powder. NMR (MeOH-d₄) δ 7.93 (m, 2H), 7.75 (m, 1H), 7.43 (m, 1H), 4.52 (m, 1H), 4.32 (m, 2H), 3.89 (m, 2H), 3.67 (m, 2H), 3.38 (m, 2H), 3.14 (m, 2H), 3.01 (s, 3H), 2.79 (m, 1H), 2.60 (m, 1H), 2.39 (m, 1H), 2.00 (m, 1H), 1.55 (s, 3H), 1.54 (s, 3H); MS (ESI+) for C₂₂H₃₀N₈O m/z 423.1 (M+H)⁺.

General Procedure L.

(1-Aminocyclohexyl)methanol

2M Lithium tetrahydroaluminate in tetrahydrofuran (80.0 mL, 160 mmol) was charged into a 500 mL 3-necked round bottomed flask (oven-dried and cooled under argon) fitted with a magnetic stir bar and the solution was cooled to 0° C. under argon. 1-Aminocyclohexanecarboxylic acid (7.64 g, 53.3 mmol) is added portionwise over a period of 1 hour. At the end of the addition, the reaction mixture was diluted with tetrahydrofuran (60 mL), slowly warmed to room temperature, and then heated at reflux for 18 hours. The mixture was cooled to room temperature. The reaction mixture was further diluted with tetrahydrofuran (160 mL) and then cooled to 0° C. Saturated aqueous sodium carbonate (100 ml) was added very slowly keeping the internal temperature below 15° C. After the addition of the carbonate solution is complete, the ice bath was left to expire and the mixture slowly warmed to room temperature overnight. The reaction mixture was filtered thru a pad of Celite® washing with ethyl acetate (400 mL). The solvent was removed in vacuo to afford a wet oil which was taken up in methylene chloride (300 mL) and dried over sodium sulfate. Filtration and concentration of the solvent in vacuo affords 6.89 g (99% yield) of the desired product as a clear colorless oil. NMR (CDCl₃) 3.34 (s, 2H), 1.81 (bs, 3H), 1.51-1.32 (m, 10H); MS (ESI+) for C₇H₁₅NO m/z 130.0 (M+H)⁺.

(1-Aminocyclopentyl)methanol

Using General Procedure L on commercially available cycloleucine affords the desired product in 99% yield as a pale yellow oil. NMR (CDCl₃) 3.40 (s, 2H), 1.86-1.61 (m, 9H), 1.46-1.29 (m, 2H); MS (ESI+) for C₆H₁₃NO m/z 116.1 (M+H)⁺.

General Procedure M.

1-{[(Triisopropylsilyl)oxy]methyl}cyclohexanamine

(1-Aminocyclohexyl)methanol (3.43 g, 26.5 mmol) was taken up in methylene chloride (80 mL) at room temperature under argon. Triethylamine (5.6 mL, 40 mmol) was added followed by the addition of triisopropylsilyl chloride (5.34 mL, 25.2 mmol). The reaction mixture was stirred at room temperature overnight during which time it became turbid. The reaction mixture was poured into a separatory funnel transferring with methylene chloride (100 mL). The organic layer was washed sequentially with water (40 mL x 2) and brine (40 mL). The organic layer was dried over sodium sulfate, filtered and the solvent concentrated in vacuo to afford 6.68 g (93% yield) of the desired product as a clear pale yellow oil. NMR (CDCl₃) δ 3.49 (s, 2H), 1.75-1.25 (m, 10H), 1.16-1.06 (m, 21H); MS (ESI+) for C₁₁H₂₇NOSi m/z 203.2 (M+H)⁺.

1-{[(Triisopropylsilyl)oxy]methyl}cyclopentanamine

Following General Procedure M and using (1-aminocyclopentyl)methanol the desired product was obtained in 85% yield as a clear dark yellow oil. NMR (CDCl₃) δ 3.53 (s, 2H), 1.85-1.39 (m, 8H), 1.16-1.07 (m, 21H); MS (ESI+) for C₁₅H₃₃NOSi m/z 272.2 (M+H)⁺.

2-[(Triisopropylsilyl)oxy]ethanamine

Following General Procedure M and using commercially available ethanolamine the desired product was obtained in 99% yield as a clear pale yellow oil. NMR (CDCl₃) δ 3.56 (t, 2H, J=6.0 Hz), 2.94 (t, 2H, J=6.0 Hz), 1.09-0.99 (m, 21H); MS (ESI+) for C₁₁H₂₇NOSi m/z 217.2 (M+H)⁺.

1-{[tert-Butyl)dimethyl)silyl]oxy}-2-methylpropan-2-amine

Following General Procedure M and using commercially available 2-amino-2-methyl-1-propanol and using tert-butyldimethylsilyl chloride the desired product was obtained in 95% yield as a clear colorless oil. NMR (CDCl₃) δ 3.31 (s, 2H), 0.93 (s, 9H), 0.06 (s, 6H); MS (ESI+) for C₁₀H₂₅NOSi m/z 204.2 (M+H)⁺.

As exemplified in Scheme 10, compounds of Formula VI can be synthesized beginning with the aldehyde illustrated above. In Step 1, an alkyne can be treated with an organic solvent, and a base optionally at a reduced temperature and subsequently treated with an aldehyde according to methods known in the art. For example, the aldehyde in Step 1 can be treated with a base, for example, isopropylmagnesium chloride lithium chloride complex in an organic solvent, for example, tetrahydrofuran at about −15° C. and next treated with an aldehyde to generate an alkyne. In Step 2, a desired alkynyl alcohol can be treated with a base in an organic solvent at an elevated temperature to isomerize the desired alkynyl alcohol to a desired alkene. For example, a desired alkynyl alcohol can be treated with a base, such as triethylamine in an organic solvent, for example, 1,4-dioxane at an elevated temperature of about 60° C. to generate an alkene. In Step 3, a desired alkene can be treated with ammonia and a mixture of organic solvents to form a tert-butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate according to methods known in the art. For example, an alkene can be treated with 0.5M ammonia and a mixture of organic solvents, for example, dioxane and acetonitrile to form a tert-butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate. In Step 4, a desired ester can be treated with a desired acid and an organic solvent to generate a desired carboxylic acid according to methods known in the art. For example, a desired ester can be treated with a desired acid, for example, trifluoroacetic acid, to generate a carboxylic acid. In one embodiment, the organic solvent is dichloromethane. In Step 5, a desired acid can be treated with a desired amine, an organic solvent and a coupling reagent to form a desired amide according to methods known in the art. For example, a desired acid can be treated with a desired amine, an organic solvent, for example, N,N-dimethylformamide, and a coupling reagent, for example, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, to generate an amide. In Step 6, a silyl protected alcohol can be treated with a fluoride reagent and an organic solvent according to methods known in the art to generate a desired alcohol. For example, a silyl protected alcohol can be treated with a fluoride reagent, for example, tetrabutylammonium fluoride, and an organic solvent, for example, acetonitrile, to generate an alcohol. In step 7, a desired alcohol can be treated with a sulfonyl chloride to generate a desired mesylate according to methods known in the art. For example, an alcohol can be treated with a desired sulfonyl chloride, for example, methanesulfonyl chloride, to generate a mesylate. In one embodiment, an amine spontaneously reacts with said mesylate to generate a cyclic amide. In Step 8, a desired thiol can be treated with a desired amine and an organic solvent at an elevated temperature to generate a desired amine according to methods known in the art. For example, a thiol can be treated with an amine, for example, 5-(4-methylpiperazin-1-yl)pyridin-2-amine, and an organic solvent, for example, N,N-dimethylacetamide, at an elevated temperature of about 150° C. to generate an amine. The compound 5-(4-methylpiperazin-1-yl)pyridin-2-amine can be prepared as disclosed in U.S. Pat. No. 8,598,186 to Tavares and Strum.

As exemplified in Scheme 11, compounds of Formula VI can be synthesized beginning with the aldehyde illustrated above. In Step 1, an alkyne can be treated with an organic solvent, and a base optionally at a reduced temperature and subsequently treated with an aldehyde according to methods known in the art. For example, the aldehyde in Step 1 can be treated with a base, for example, isopropylmagnesium chloride lithium chloride complex in an organic solvent, for example, tetrahydrofuran at about −15° C. and next treated with an aldehyde to generate an alkyne. In Step 2, a desired alkynyl alcohol can be treated with a base in an organic solvent at an elevated temperature to isomerize the desired alkynyl alcohol to a desired alkene. For example, a desired alkynyl alcohol can be treated with a base, such as triethylamine in an organic solvent, for example, 1,4-dioxane at an elevated temperature of about 60° C. to generate an alkene. In Step 3, a desired alkene can be treated with ammonia and a mixture of organic solvents to form a tert-butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate according to methods known in the art. For example, an alkene can be treated with 0.5M ammonia and a mixture of organic solvents, for example, dioxane and acetonitrile to form a tert-butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate. In Step 4, an amine can be treated with a base, an organic solvent, and a cyclic sulfamidate to form an amine according to methods known in the art. For example, a desired amine can be treated with a base, for example, triethylamine, an organic solvent, for example, N,N-dimethylformamide, and a cyclic sufamidate to form an amine. In Step 5, a protected amine can be treated with an organic acid to form an amine that can subsequently form a cyclic amide according to methods known in the art. For example, a protected amine can be treated with an organic acid, for example, trifluoroacetic acid, and subsequently react with an ester to form a cyclic amide.

EXAMPLES

The patents WO 2013/148748 entitled “Lactam Kinase Inhibitors” to Tavares, F. X., WO 2013/163239 entitled “Synthesis of Lactams” to Tavares, F. X., and U.S. Pat. No. 8,598,186 entitled “CDK Inhibitors” to Tavares, F. X. and Strum, J. C. are incorporated by reference herein in their entirety.

Example 1 Synthesis of Compound 2 (Scheme 1)

Compound 2 is synthesized according to the method of A. Haidle et al., See, WO 2009/152027 entitled “5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives for MARK inhibition.”

Example 2 Synthesis of Compound 3 (Scheme 1)

Step 1: A round-bottomed flask inserted with a nitrogen atmosphere is charged with Compound 2, ethanol, and lithium borohydride at ambient temperature. The reaction is stirred at ambient temperature and monitored by thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Once Compound 2 can no longer be detected, the reaction is quenched with an aqueous acid such as aqueous hydrochloric acid, diluted with ethyl acetate and the layers separated. The organic layer is dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The product, a primary alcohol, is purified by silica gel column chromatography eluting with a hexane-ethyl acetate gradient and used directly in the next step.

Step 2: A round-bottomed flask inserted with a nitrogen atmosphere is charged with the primary alcohol prepared in step 1, DMF and phosphorus tribromide. The reaction is stirred at ambient temperature and monitored by thin layer chromatography (TLC) or HPLC. Once the primary alcohol can no longer be detected, the reaction is quenched with brine and diluted with toluene. The layers are separated and the toluene layer is dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The bromide is purified by silica gel column chromatography eluting with a hexane—ethyl acetate gradient.

Example 3 Synthesis of Compound 5 (Scheme 1)

A round-bottomed flask inserted with a nitrogen atmosphere is charged with tetrahydrofuran and the lactam 4, described below. The reaction is cooled to −78° C. and lithium diisopropylamide solution (2M in THF/heptane/ethyl benzene) is added dropwise. To the resulting enolate is added Compound 3, dropwise, and the reaction is allowed to warm to room temperature overnight. The reaction is diluted with saturated brine and the layers are separated. The organic layer is dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a dichloromethane -methanol gradient.

Example 4 Synthesis of Compound 6 (Scheme 1)

A round-bottomed flask is charged with Compound 5 and an aqueous acid, for example a pH=1 HCl solution. The reaction is allowed to stir at room temperature until starting material is no longer detected by thin layer chromatography or HPLC. The reaction is neutralized with solid K₂CO₃ and diluted with dichloromethane. The layers are separated, the organic layer dried over anhydrous magnesium sulfate, filtered and concentrated. Compound 6 is purified by silica gel column chromatography eluting with a dichloromethane—methanol gradient.

Example 5 Synthesis of Compound 7 (Scheme 1)

A round-bottomed flask inserted with a nitrogen atmosphere is charged with Compound 6, ethanol and DBU (10 eq). The reaction is monitored by thin layer chromatography or HPLC. Note: The reaction can be heated at reflux if necessary. Once Compound 6 is no longer detected, the reaction is concentrated in vacuo. The lactam 7 is purified by silica gel column chromatography eluting with a dichloromethane—methanol gradient.

Example 6 Synthesis of Compound 8 (Scheme 1)

A round-bottomed flask inserted with a nitrogen atmosphere is charged with Compound 7, meta-chloroperoxybenzoic acid, an organic solvent and stirred at ambient temperature. The reaction is monitored by thin layer chromatography or HPLC. Once Compound 7 is no longer detected, the reaction is concentrated in vacuo. Compound 8 is purified by silica gel column chromatography eluting with a dichloromethane—methanol gradient.

Example 7 Synthesis of Compound 10 (Scheme 1)

The tricyclic lactam 8 is combined with an amine (9, 0.9 eq) and an organic solvent such as tetrahydrofuran. A strong base such as lithium hexamethyldisilazane is added and the reaction is stirred until lactam 8 is no longer detected by either thin layer chromatography or HPLC. The reaction is concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a dichloromethane—methanol gradient.

Alternatively, a CEM Discovery microwave vessel is charged with the tricyclic lactam 8, N-methyl-2-pyrrolidone (NMP), Hunig's base, and amine 9 (0.9 eq). The reaction is heated at 150° C. for 1-4 hours while being monitored by TLC. Once the tricyclic lactam 8 is no longer detected by TLC or HPLC, the reaction is concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a dichloromethane—methanol gradient.

Example 8 Synthesis of Compound 11 (Scheme 2)

Compound 7 is treated with an oxidizing agent such as 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in an organic solvent to generate the alkene intermediate 12.

Example 9 Synthesis of Compound 14 (Scheme 2)

The sulfone intermediate 12 is combined with an amine (13, 0.9 eq) in an organic solvent such as tetrahydrofuran. An organic base such as lithium hexamethyldisilazane is added and the reaction is stirred until sulfone intermediate 12 can no longer be detected by thin layer chromatography or HPLC. The product is purified by silica gel column chromatography eluting with a dichloromethane—methanol gradient.

Alternatively, a CEM Discovery microwave vessel is charged with the sulfone intermediate 12, N-methyl-2-pyrrolidone (NMP), Hunig's base, and amine 13 (0.9 eq). The reaction is heated at 150° C. for 1-4 hours while being monitored by TLC. Once the sulfone intermediate 12 is no longer detected by TLC or HPLC, the reaction is concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a dichloromethane -methanol gradient.

Example 10 Synthesis of Compound 4

Step 1: Synthesis of Compound 15 (Scheme 3)

Compound 15 is synthesized according to the method of Arigon, J., See, US 2013/0289031, entitled “Pyrimidinone derivatives, preparation thereof and pharmaceutical use thereof”

Step 2: Synthesis of Compound 16 (Scheme 3)

A round-bottomed flask inserted with a nitrogen atmosphere is charged with Compound 15, dichloromethane and triethylamine (1.5 eq). The reaction is cooled to 0° C. and Boc anhydride (1.5 eq) is added. The reaction is allowed to stir at room temperature until Compound 15 is no longer detected by thin layer chromatography or HPLC. The reaction is concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a hexane—ethyl acetate gradient.

Step 3: Synthesis of Compound 17 (Scheme 3)

A round-bottomed flask inserted with a nitrogen atmosphere is charged with Compound 16, acetonitrile and a base such as potassium carbonate. Methyl chloroacetate is added dropwise. The reaction is allowed to stir at room temperature until Compound 16 is no longer detected by thin layer chromatography or HPLC. The reaction is concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a hexane—ethyl acetate gradient.

Step 4: Synthesis of Compound 18 (Scheme 3)

Compound 17 is dissolved in a solution comprising 3M HCl in methanol and the reaction is stirred at ambient temperature. Note: the reaction can be heated at a temperature of about 25° C. to about 60° C. to accelerate the reaction rate. Once the starting material is no longer detected by thin layer chromatography, the reaction is concentrated in vacuo. The product is purified by silica gel column chromatography using a dichloromethane—methanol gradient

Step 5: Synthesis of Compound 19 (Scheme 3)

A round-bottomed flask inserted with a nitrogen atmosphere is charged with Compound 18, dichloromethane, and diisopropylethylamine (1.2 eq). Chloromethyl methyl ether (MOM-Cl, 1.2 eq) is added dropwise. The reaction is allowed to stir at room temperature and monitored by TLC. Once the starting material is no longer detected by thin layer chromatography, the reaction is quenched with saturated brine solution. The organic layer is separated, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The product is purified by silica gel column chromatography using a dichloromethane—methanol gradient.

Step 6: Synthesis of Compound 4

A round-bottomed flask inserted with a nitrogen atmosphere is charged with anhydrous tetrahydrofuran and Compound 19. The reaction is cooled to −78° C. Sodium bis(trimethylsilyl)amide (1M in THF, 1.1 eq) is added dropwise. Chloromethyl methyl ether (MOM-Cl, 1.2 eq) is added dropwise with stirring and the reaction is allowed to warm to room temperature overnight. The reaction is quenched with saturated brine solution and the layers are separated. The organic layer is dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The product is purified by silica gel column chromatography using a dichloromethane—methanol gradient.

Example 11 Synthesis of Compound 25 (Scheme 5)

Compound 20 is commercially available. Compound 25 is synthesized according to the synthetic methodology disclosed in Example 10.

Example 12 Synthesis of Compound 31 (Scheme 6)

Compound 31 is synthesized according to the synthetic methodology disclosed in Example 10.

Example 13 Synthesis of Compound 33 (Scheme 7)

Step 1: Synthesis of Compound 32

Compound 32, 5-morpholinopyrid-2-amine, is synthesized according to Tavares, F. X. and Strum, J. C., See, U.S. Pat. No. 8,598,186, entitled “CDK Inhibitors”.

Step 2: Synthesis of Compound 33

The sulfone intermediate 8 is diluted with a suitable solvent such as tetrahydrofuran and an organic base such as lithium hexamethyldisilazane is added. The amine 32 is added and the reaction is stirred until sulfone intermediate 8 can no longer be detected by thin layer chromatography or HPLC. The product is purified by silica gel column chromatography eluting with a dichloromethane—methanol gradient.

Alternatively, a CEM Discovery microwave vessel is charged with the sulfone intermediate 8, N-methyl-2-pyrrolidone (NMP), Hunig's base, and 5-morpholinopyrid-2-amine (0.9 eq). The reaction is heated at 150° C. for 1-4 hours while being monitored by TLC. Once the sulfone intermediate 8 is no longer detected by TLC or HPLC, the reaction is concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a dichloromethane—methanol gradient.

Example 14 Synthesis of Compound 34 (Scheme 8)

Step 1: Synthesis of Compound 32

Compound 32, 5-morpholinopyrid-2-amine, is synthesized according to Tavares, F. X. and Strum, J. C., See, U.S. Pat. No. 8,598,186, entitled “CDK Inhibitors”.

Step 2: Synthesis of Compound 34

The sulfone intermediate 12 is combined with a suitable solvent such as tetrahydrofuran and an organic base such as lithium hexamethyldisilazane. The amine 32 is added and the reaction is stirred until sulfone intermediate 12 can no longer be detected by thin layer chromatography or HPLC. The product is purified by silica gel column chromatography eluting with a dichloromethane—methanol gradient.

Alternatively, a CEM Discovery microwave vessel is charged with the sulfone intermediate 12, N-methyl-2-pyrrolidone (NMP), Hunig's base, and 5-morpholinopyrid-2-amine (0.9 eq). The reaction is heated at 150° C. for 1-4 hours while being monitored by TLC. Once the sulfone intermediate 12 is no longer detected by TLC or HPLC, the reaction is concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a dichloromethane—methanol gradient.

Example 15 Preparation of a Formula V compound

Step 1: Compound 7 is Boc protected according to the method of A. Sarkar et al. (JOC, 2011, 76, 7132-7140).

Step 2: Boc-protected Compound 7 is treated with 5 mol % NiCl₂(Ph₃)₂, 0.1 eq triphenylphosphine, 3 eq Mn, 0.1 eq tetraethylammonium iodide, in DMI under CO₂ (1 atm) at 25° C. for 20 hours to convert the methyl thiol derivative into the carboxylic acid.

Step 3: The carboxylic acid from Step 2 is converted to the corresponding acid chloride using standard conditions.

Step 4: The acid chloride from Step 3 is reacted with N-methyl piperazine to generate the corresponding amide.

Step 5: The amide from Step 4 is deprotected using trifluoroacetic acid in methylene chloride to generate the target compound. The product is purified by silica gel column chromatography eluting with a dichloromethane—methanol gradient.

Example 16 CDK4/6 Inhibition In Vitro Assay

Selected compounds disclosed herein were tested in CDK4/cyclinD1, CDK6/CycD3, CDK2/CycA, CDK2/cyclinE, CDK5/p25, CDK5/p35, CDK7/CycH/MAT1, and CDK9/CycT kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Phosphoacceptor substrate peptide concentration used was 1.25 μM for all assays (except μM 10 was used for the CKD7/CycH/MAT1 assay and Staurosporine was used as the reference compound for all assays. Specifics of each assay are as described below:

CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM; Incubation time: 3 hr.

CDK2/CyclinE: Enzyme concentration: 0.2 nM; ATP concentration: 100 μM; Incubation time: 3 hr.

CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr.

CDK6/CyclinD3: Enzyme concentration: 10 nM; ATP concentration: 300 μM; Incubation time: 3 hr.

CDK5/p25: Enzyme concentration: 0.1 nM; ATP concentration: 20 μM; Incubation time: 3 hr.

CDK5/p35: Enzyme concentration: 0.07 nM; ATP concentration: 20 μM; Incubation time: 3 hr.

CDK7/CycH/MAT1: Enzyme concentration: 5 nM; ATP concentration: 50 μM; Incubation time: 3 hr.

CDK9/CycT: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.

TABLE 2 Inhibition of CDK kinases by Tricyclic Lactam Compounds Cdk7/ Compound Cdk2/ Cdk2/ Cdk4/ Cdk5/ Cdk5/ Cdk6/ CycH/ Cdk9/ No. CycA CycE CycD1 p25 p35 CycD3 MAT1 Cyc T ZZZ * * * * * * * YYY * * *** * * ** * BBBB ** * ** * * ** * * AAAA * * * * * * * * CCCC * * * * * * * * GGGG * * ** * * ** * * * >100 μM ** 10 μM < X > 100 μM *** <10 μM

Example 17 G1 Arrest (Cellular G1 and S-phase) Assay

For determination of cellular fractions in various stages of the cell cycle following various treatments, HS68 cells (human skin fibroblast cell line (Rb-positive)) are stained with propidium iodide staining solution and run on Dako Cyan Flow Cytometer. The fraction of cells in G0-G1 DNA cell cycle versus the fraction in S-phase DNA cell cycle is determined using FlowJo 7.2.2 analysis.

Example 18 Inhibition of Cellular Proliferation

Cellular proliferation assays are conducted using the following cancer cell lines: MCF7 (breast adenocarcinoma—Rb-positive), ZR-75-1 (breast ductal carcinoma—Rb-positive), H69 (human small cell lung cancer—Rb-negative) cells, or A2058 (human metastatic melanoma cells—Rb-negative). These cells are seeded in Costar (Tewksbury, Mass.) 3093 96 well tissue culture treated white walled/clear bottom plates. Cells are treated with the compounds of Table 1 as nine point dose response dilution series from 10 uM to 1 nM. Cells are exposed to compounds and then cell viability is determined after either four (H69) or six (MCF7, ZR75-1, A2058) days as indicated using the CellTiter-Glo® luminescent cell viability assay (CTG; Promega, Madison, Wis., United States of America) following the manufacturer's recommendations. Plates are read on BioTek (Winooski, Vt.) Syngergy2 multi-mode plate reader. The Relative Light Units (RLU) are plotted as a result of variable molar concentration and data is analyzed using Graphpad (LaJolla, Californaia) Prism 5 statistical software to determine the EC₅₀ for each compound.

Example 19 Pharmacokinetic and Pharmacodynamic Properties of Tricyclic Lactam Compounds

Tricyclic lactam compounds are dosed to mice at 30 mg/kg by oral gavage or 10 mg/kg by intravenous injection. Blood samples are taken at 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 hours post dosing and the plasma concentration of compounds are determined by HPLC.

Example 20 Cellular Wash-Out Experiment

HS68 cells are seeded out at 40,000 cells/well in 60 mm dish on day 1 in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin/streptomycin and 1× Glutamax (Invitrogen) as described (Brookes et al. EMBO J, 21(12)2936-2945 (2002) and Ruas et al. Mol Cell Biol, 27(12)4273-4282 (2007)). 24 hrs post seeding, cells are treated with a tricyclic lactam compound or DMSO vehicle alone at 300 nM final concentration of test compounds. On day 3, one set of treated cell samples are harvested in triplicate (0 Hour sample). Remaining cells are washed two times in PBS-CMF and returned to culture media lacking test compound. Sets of samples were harvested in triplicate at 24, 40, and 48 hours.

Alternatively, the same experiment is done using normal Renal Proximal Tubule Epithelial Cells (Rb-positive) obtained from American Type Culture Collection (ATCC, Manassas, Va.). Cells are grown in an incubator at 37° C. in a humidified atmosphere of 5% CO₂ in Renal Epithelial Cell Basal Media (ATCC) supplemented with Renal Epithelial Cell Growth Kit (ATCC) in a 37° C. humidified incubator.

Upon harvesting cells, samples are stained with propidium iodide staining solution and samples run on Dako Cyan Flow Cytometer. The fraction of cells in G0-G1 DNA cell cycle versus the fraction in S-phase DNA cell cycle is determined using FlowJo 7.2.2 analysis.

Example 21 Bone Marrow Proliferation as Evaluated Using EdU Incorporation and Flow Cytometry Analysis

For hematopoietic stem cell and/or hematopoietic progenitor cell (HSPC) proliferation experiments, young adult female FVB/N mice are dosed with a single dose of compound by oral gavage. Mice are then sacrificed at 0, 12, 24, 36, or 48 hours following compound administration, and bone marrow is harvested, as previously described (Johnson et al. J. Clin. Invest. (2010) 120(7), 2528-2536). Four hours before the bone marrow is harvested, mice are treated with 100 μg of EdU by intraperitoneal injection (Invitrogen). Bone marrow mononuclear cells are harvested and immunophenotyped using previously described methods and percent EdU positive cells are then determined (Johnson et al. J. Clin. Invest. (2010) 120(7), 2528-2536). In brief, HSPCs are identified by expression of lineage markers (Lin−), Sca1 (S+), and c-Kit (K+).

Example 22 Metabolic Stability

The metabolic stability of tricyclic lactam compounds can be determined in human, dog, rat, monkey, and mouse liver microsomes. Human, mouse, and dog liver microsomes are purchased from Xenotech, and Sprague-Dawley rat liver microsomes are prepared by Absorption Systems. The reaction mixture comprising 0.5 mg/mL of liver microsomes, 100 mM of potassium phosphate, pH 7.4, 5 mM of magnesium chloride, and 1 uM of test compound is prepared. The test compound is added into the reaction mixture at a final concentration of 1 uM. An aliquot of the reaction mixture (without cofactor) is incubated in a shaking water bath at 37° C. for 3 minutes. The control compound, testosterone, is run simultaneously with the test compound in a separate reaction. The reaction is initiated by the addition of cofactor (NADPH), and the mixture is then incubated in a shaking water bath at 37° C. Aliquots (100 μL) are withdrawn at 0, 10, 20, 30, and 60 minutes for the test compound and 0, 10, 30, and 60 minutes for testosterone. Test compound samples are immediately combined with 100 μL of ice-cold acetonitrile containing internal standard to terminate the reaction. Testosterone samples are immediately combined with 800 μL of ice cold 50/50 acetonitrile/dH₂O containing 0.1% formic acid and internal standard to terminate the reaction. The samples are assayed using a validated LC-MS/MS method. Test compound samples are analyzed using the Orbitrap high resolution mass spectrometer to quantify the disappearance of parent test compound and detect the appearance of metabolites. The peak area response ration (PARR) to internal standard is compared to the PARR at time 0 to determine the percent of test compound or positive control remaining at time-point. Half-lives are calculated using GraphPad software, fitting to a single-phase exponential decay equation. Half-life is calculated based on t½=0.693 k, where k is the elimination rate constant based on the slope plot of natural logarithm percent remaining versus incubation time.

Example 23 Efficacy of Tricyclic Lactams in HER2-Driven Breast Tumors

A HER2-driven model (Rb-positive) of breast cancer (Muller W J, Sinn E, Pattengale P K, Wallace R, Leder P. Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene. Cell 1988; 54: 105-15), that expresses c-neu (the mouse ortholog of human HER2) driven by the MMTV promoter is used in the following example. This model is chosen because previous studies in murine (Yu Q, Geng Y, Sicinski P. Specific protection against breast cancers by cyclin D1 ablation. Nature 2001; 411: 1017-21; Landis M W, Pawlyk B S, Li T, Sicinski P, Hinds P W. Cyclin D1-dependent kinase activity in murine development and mammary tumorigenesis. Cancer Cell 2006; 9: 13-22; Reddy H K, Mettus R V, Rane S G, Grana X, Litvin J, Reddy E P. Cyclin-dependent kinase 4 expression is essential for neu-induced breast tumorigenesis. Cancer Res 2005; 65: 10174-8; Yu Q, Sicinska E, Geng Y, Ahnstrom M, Zagozdzon A, Kong Y, et al. Requirement for CDK4 kinase function in breast cancer. Cancer Cell 2006; 9: 23-32.) and human HER2-positive breast cancer (An H X, Beckmann M W, Reifenberger G, Bender H G, Niederacher D. Gene amplification and overexpression of CDK4 in sporadic breast carcinomas is associated with high tumor cell proliferation. Am J Pathol 1999; 154: 113-8; Samady L, Dennis J, Budhram-Mahadeo V, Latchman D S. Activation of CDK4 gene expression in human breast cancer cells by the Brn-3b POU family transcription factor. Cancer Biol Ther 2004; 3: 317-23; Takano Y, Takenaka H, Kato Y, Masuda M, Mikami T, Saegusa M, et al. Cyclin D1 overexpression in invasive breast cancers: correlation with cyclin-dependent kinase 4 and oestrogen receptor overexpression, and lack of correlation with mitotic activity. J Cancer Res Clin Oncol 1999; 125: 505-12) suggest that these tumors require CDK4/6 and CCND1 for progression and maintenance.

MMTV-neu mice are generated and observed post-lactation, with tumors observed with a median latency of approximately 25 weeks. Mice are enrolled in therapy studies when tumors reach a standard size (50-60 mm3) that permitted easy serial assessment. Tumor-bearing mice are continuously treated with a tricyclic lactam compound added to their chow (100 mg/kg/d or 150 mg/kg/d). MMTV-c-neu mice are examined weekly to assess tumor development by palpation. Tumor volumes are calculated by the formula, Volume=[(width)²×length]/2. Tumor-bearing mice are euthanized at the indicated times due to predefined morbidity, tumor ulceration, or a tumor size of more than 1.5 cm in diameter.

Example 24 Efficacy of Tricyclic Lactams in HER2-Driven Breast Tumors

The in vivo efficacy of the tricyclic lactams is tested in the genetically engineered mouse model of luminal breast cancer. Tumors are serially assessed weekly using caliper measurements. Therapeutic intervention begins once tumors reach 40-64 mm³. Tumor volume is calculated using the formula ((Width)×Length)/2. Compounds are administered orally via medicated diets (100 mg/kg/d). Medicated diets are administered for 28 consecutive days and then stopped. RECIST criteria are used to assess objective response rates.

Example 25 Cell Cycle Arrest by Tricyclic Lactams in CDK4/6-Dependent Cells

To test the ability of tricyclic lactams to induce a clean G1-arrest, a cell based screening method is used consisting of two CDK4/6-dependent cell lines (tHS68 and WM2664; Rb-positive) and one CDK4/6-independent (A2058; Rb-negative) cell line. Twenty-four hours after plating, each cell line is treated with a tricyclic lactam compound in a dose dependent manner for 24 hours. At the conclusion of the experiment, cells are harvested, fixed, and stained with propidium iodide (a DNA intercalator), which fluoresces strongly red (emission maximum 637 nm) when excited by 488 nm light. Samples are run on a Dako Cyan flow cytometer and >10,000 events are collected for each sample. Data are analyzed using FlowJo 2.2 software developed by TreeStar, Inc.

Example 26 Inhibition of RB Phosphorylation

The CDK4/6-cyclin D complex is essential for progression from G1 to the S-phase of the DNA cell cycle. This complex phosphorylates the retinoblastoma tumor suppressor protein (Rb). To demonstrate the impact of tricyclic lactams on Rb phosphorylation (pRb), compounds are exposed to three cell lines, two CDK4/6 dependent (tHS68, WM2664; Rb-positive) and one CDK4/6 independent (A2058; Rb-negative). Twenty four hours after seeding, cells are treated with a tricyclic lactam compound at 300 nM final concentration for 4, 8, 16, and 24 hours. Samples are lysed and protein is assayed by western blot analysis. Rb phosphorylation is measured at two sites targeted by the CDK4/6-cyclin D complex, Ser780 and Ser807/811 using species specific antibodies.

Example 27 Preparation of Drug Product

The active compounds of the present invention can be prepared for intravenous administration using the following procedure. The excipients hydroxypropyl-beta-cyclodextrin and dextrose can be added to 90% of the batch volume of USP Sterile Water for Injection or Irrigation with stirring; stir until dissolved. The active compound in the hydrochloride salt form is added and stirred until it is dissolved. The pH is adjusted with 1N NaOH to pH 4.3+0.1 and 1N HCl can be used to back titrate if necessary. USP sterile water for injection or irrigation can be used to bring the solution to the final batch weight. The pH is next re-checked to ensure that the pH is pH 4.3+0.1. If the pH is outside of the range add 1N HCl or 1N NaOH as appropriate to bring the pH to 4.3+0.1. The solution is next sterile filtered to fill 50 or 100 mL flint glass vials, stopper, and crimped.

This specification has been described with reference to embodiments of the invention. The invention has been described with reference to assorted embodiments, which are illustrated by the accompanying Examples. The invention can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Given the teaching herein, one of ordinary skill in the art will be able to modify the invention for a desired purpose and such variations are considered within the scope of the invention. 

We claim:
 1. A compound of Formula VI:

wherein Z is —(CH₂)_(x)— wherein x is 1, 2, 3, or 4 or —O—(CH₂)_(z)— wherein z is 2, 3, or 4; each X is independently CH or N; R is H, C₁-C₃ alkyl or haloalkyl, cycloalkyl, cycloalkyl containing one or more heteroatoms selected from N, O, or S, -(alkylene)_(m)C₃-C₈ cycloalkyl, -(alkylene)_(m)-aryl, -(alkylene)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)-NR³R⁴, -(alkylene)_(m)—O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴; each R¹ is independently aryl, alkyl, cycloalkyl, or haloalkyl, wherein each of said alkyl, cycloalkyl, and haloalkyl groups optionally includes O or N heteroatoms in place of a carbon in the chain and two R¹s on adjacent ring atoms or on the same ring atom together with the ring atom(s) to which they are attached optionally form a 3-8-membered cycle; y is 0, 1, 2, 3, or 4; R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴, -(alkylene)_(m)-C(O)—O-alkyl, -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)-R⁵, or -(alkylene)_(m)-S(O)_(n)-NR³R⁴ any of which may be optionally independently substituted with one or more R^(x) groups as allowed by valance; R³ and R⁴ at each occurrence are independently: (i) hydrogen or (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl; or R³ and R⁴ together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring; R⁵ is: (i) hydrogen or (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarvlalkyl; R^(x) at each occurrence is independently, halo, cyano, nitro, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, or heterocycloalkyl; n is 0, 1, or 2; m is 0, 1, or 2; R⁶ is H, lower alkyl, -(alkylene)m-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)-NR³R⁴, -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)n-R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴; each R¹⁴ is independently H, C₁-C₃ alkyl or haloalkyl, cycloalkyl, cycloalkyl containing one or more heteroatoms selected from N, O, or S, -(alkylene)_(m)-C₃-C₈cycloalkyl, -(alkylene)m-aryl, -(alkylene)m-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴, -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴; or two R¹⁴ groups bonded to the same carbon form an exocyclic double bond; or two R¹⁴ groups bonded to the same carbon form a carbonyl group; and when the compound of Formula VI has a double bond, as indicated by the (----), in the 6-membered ring fused to the pyrimidine ring, two R¹⁴ groups are present as allowed for in Formula VI above; or when the compound of Formula VI does not include a double bond, as indicated by the (----), in the 6-membered ring fused to the pyrimidine ring, four R¹⁴ groups are present as allowed for in Formula VI above; or a pharmaceutically acceptable salt thereof.
 2. A compound of claim 1, wherein the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 3. A compound of Formula VI:

wherein R, R¹, R², R³, R⁴, R⁵, R⁶, R^(x), Z, m, n, and y are as defined in claim 1; each R¹⁴ is independently H, C₁-C₃ alkyl or haloalkyl, cycloalkyl, cycloalkyl containing one or more heteroatoms selected from N, O, or S, -(alkylene)_(m)-C₃-C₈cycloalkyl, -(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)- heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴, -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴; or two R¹⁴ groups bonded to the same carbon form an exocyclic double bond; or two R¹⁴ groups bonded to the same carbon form a carbonyl group; and when the compound of Formula VI has a double bond, as indicated by the (----), in the6-membered ring fused to the pyrimidine ring, two R¹⁴ groups are present as allowed for in Formula VI above; or when the compound of Formula VI does not include a double bond, as indicated by the (----), in the 6-membered ring fused to the pyrimidine ring, four R¹⁴ groups are present as allowed for in Formula VI above; wherein each heteroaryl is an aryl ring system that contains one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized; wherein each aryl is a carbocyclic aromatic system containing one or two rings, wherein such rings may be attached together in a fused manner, and wherein each aryl may have 1 or more R^(x) sub stituents; wherein each heterocyclo is a saturated or partially saturated heteroatom-containing ring radical, where the heteroatoms may be selected from nitrogen, sulfur and oxygen, wherein each heterocyclo is a monocyclic 6-8 membered ring or a 5-16 membered bicyclic ring system, and wherein each heterocyclo may have 1 to 3 R^(x) substituents; or a pharmaceutically acceptable salt thereof.
 4. A compound having the structure:


5. The compound of claim 1, wherein the compound is:


6. The compound of claim 1, wherein the compound is:


7. The compound of claim 1, wherein the compound is:


8. The compound of claim 1, wherein the compound is:


9. The compound of claim 1, wherein the compound is:


10. The compound of claim 1, wherein R is H.
 11. The compound of claim 3, wherein R is H.
 12. The compound of claim 10, wherein two R¹⁴ groups bonded to the same carbon form an exocyclic double bond, or two R¹⁴ groups bonded to the same carbon form a carbonyl group.
 13. The compound of claim 11, wherein two R¹⁴ groups bonded to the same carbon form an exocyclic double bond, or two R¹⁴ groups bonded to the same carbon form a carbonyl group.
 14. The compound of claim 12, wherein one X is N and the other X is CH.
 15. The compound of claim 13, wherein one X is N and the other X is CH.
 16. The compound of claim 14, wherein R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴, -(alkylene)_(m)-C(O)—O-alkyl, or -(alkylene)_(m)-O—R⁵.
 17. The compound of claim 15, wherein R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴, -(alkylene)_(m)-C(O)—O-alkyl, or -(alkylene)_(m)-O—R⁵.
 18. The compound of claim 16, wherein m is
 0. 19. The compound of claim 17, wherein m is
 0. 20. The compound of claim 14, wherein R² is -(alkylene)_(m)-heterocyclo, or -(alkylene)_(m)-heteroaryl.
 21. The compound of claim 15, wherein R² is -(alkylene)_(m)-heterocyclo, or -(alkylene)_(m)-heteroaryl.
 22. The compound of claim 20, wherein heterocyclo is piperazine, piperidine, morpholine, pyrolidine, azetidine, or oxetane.
 23. The compound of claim 21, wherein heterocyclo is piperazine, piperidine, morpholine, pyrolidine, azetidine, or oxetane.
 24. The compound of claim 20, wherein heterocyclo is piperazine.
 25. The compound of claim 21, wherein heterocyclo is piperazine.
 26. The compound of claim 24, wherein R^(x) is alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, or heterocycloalkyl.
 27. The compound of claim 25, wherein R^(x) is alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, or heterocycloalkyl.
 28. The compound of claim 24, wherein R^(x) is alkyl, cycloalkyl, heterocyclo, aryl, or heteroaryl.
 29. The compound of claim 25, wherein R^(x) is alkyl, cycloalkyl, heterocyclo, aryl, or heteroaryl.
 30. The compound of claim 24, wherein R^(x) is alkyl.
 31. The compound of claim 25, wherein R^(x) is alkyl.
 32. The compound of claim 26, wherein Z is -(CH₂)_(x)—.
 33. The compound of claim 32, wherein x is 1 or
 2. 34. The compound of claim 33, wherein y is
 2. 35. The compound of claim 34, wherein R¹ is aryl, alkyl, or cycloalkyl.
 36. The compound of claim 34, wherein R¹ is alkyl.
 37. The compound of claim 34, wherein two R¹s on the same ring atom together with the ring atom to which they are attached form a 3-8-membered cycle.
 38. The compound of claim 33, wherein y is
 0. 39. The compound of claim 33, wherein y is
 1. 40. The compound of claim 1, wherein two R¹⁴ groups bonded to the same carbon form an exocyclic double bond, or two R¹⁴ groups bonded to the same carbon form a carbonyl group.
 41. The compound of claim 1, wherein one X is N and the other X is CH.
 42. The compound of claim 1, wherein R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴, -(alkylene)_(m)-C(O)—O-alkyl, or -(alkylene)_(m)-O—R⁵.
 43. The compound of claim 1, wherein m is
 0. 44. The compound of claim 1, wherein R² is -(alkylene)_(m)-heterocyclo, or -(alkylene)_(m)-heteroaryl.
 45. The compound of claim 1, wherein heterocyclo is piperazine, piperidine, morpholine, pyrolidine, azetidine, or oxetane.
 46. The compound of claim 1, wherein heterocyclo is piperazine.
 47. The compound of claim 1, wherein R^(x) is alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, or heterocycloalkyl.
 48. The compound of claim 1, wherein R^(x) is alkyl, cycloalkyl, heterocyclo, aryl, or heteroaryl.
 49. The compound of claim 1, wherein R^(x) is alkyl.
 50. The compound of claim 1, wherein Z is -(CH₂)_(x)—.
 51. The compound of claim 1, wherein x is 1 or
 2. 52. The compound of claim 1, wherein y is
 2. 53. The compound of claim 1, wherein R¹ is aryl, alkyl, or cycloalkyl.
 54. The compound of claim 1, wherein R¹ is alkyl.
 55. The compound of claim 1, wherein two R¹s on the same ring atom together with the ring atom to which they are attached form a 3-8-membered cycle.
 56. The compound of claim 1, wherein y is
 0. 57. The compound of claim 1, wherein y is
 1. 58. A compound of Formula VI:

wherein Z is -(CH₂), wherein x is 1, 2, 3, or 4 or —O—(CH₂)_(z)— wherein z is 2, 3, or 4; each X is independently CH or N; R is H, C₁-C₃ alkyl or haloalkyl, cycloalkyl, cycloalkyl containing one or more heteroatoms selected from N, O, or S, -(alkylene)_(m)-C₃-C₈cycloalkyl, -(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)m-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴, -(alkylene)_(m)-O—R⁵, -(alkylene)m-S(O)_(n)-R⁵, or -(alkylene)_(m)-S(O)_(n)-NR³R⁴; each R¹ is independently aryl, alkyl, cycloalkyl, or haloalkyl, wherein each of said alkyl, cycloalkyl, and haloalkyl groups optionally includes O or N heteroatoms in place of a carbon in the chain and two R¹s on adjacent ring atoms or on the same ring atom together with the ring atom(s) to which they are attached optionally form a 3-8-membered cycle; y is 0, 1, 2, 3, or 4; R² is

R^(2*) is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-, -(alkylene) _(m)-C(O)-(alkylene) _(m)-, -(alkylene) _(m)-S(O)₂-(alkylene) _(m)-, or -(alkylene) _(m)-NH-(alkylene) _(m)-, P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group; each R^(x1) is independently alkyl, —(C(O))—O-alkyl, -(alkylene)m-cycloalkyl, or -(alkylene) _(m)-heterocyclyl; t is 0, 1, or 2; R³ and R⁴ at each occurrence are independently: (i) hydrogen or (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl; or R³ and R⁴ together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring; R⁵ is: (i) hydrogen or (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl; n is 0, 1, or 2; m is 0, 1, or 2; R⁶ is H, lower alkyl, -(alkylene) _(m)-heterocyclo, -(alkylene) _(m)-heteroaryl, -(alkylene) _(m)-NR³R⁴, -(alkylene) _(m)-C(O)—NR³R⁴, -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)-R⁵, or -(alkylene)_(m)-S(O)n-NR³R⁴; each R¹⁴ is independently H, C₁-C₃ alkyl or haloalkyl, cycloalkyl, cycloalkyl containing one or more heteroatoms selected from N, O, or S, -(alkylene) _(m)- C₃-C₈ cycloalkyl, -(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴, -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)-R⁵, or -(alkylene) _(m)-S(O)_(n)-NR³R⁴; or two R¹⁴ groups bonded to the same carbon form an exocyclic double bond; or two R¹⁴ groups bonded to the same carbon form a carbonyl group; and when the compound of Formula VI has a double bond, as indicated by the (----), in the 6-membered ring fused to the pyrimidine ring, two R¹⁴ groups are present as allowed for in Formula VI above; or when the compound of Formula VI does not include a double bond, as indicated by the (----), in the 6-membered ring fused to the pyrimidine ring, four R¹⁴ groups are present as allowed for in Formula VI above; or a pharmaceutically acceptable salt thereof.
 59. The compound of claim 58, wherein

is


60. The compound of claim 58, wherein

is 